A Proteome-Centric View of Ageing, including that of the Skin and Age-Related Diseases: Considerations of a Common Cause and Common Preventative and Curative Interventions.

The proteome comprises all proteins of a cell or organism. To carry their catalytic and structure-related functions, proteins must be correctly folded into their unique native three-dimensional structures. Common oxidative protein damage affects their functionality by impairing their catalytic and interactive specificities. Oxidative damage occurs preferentially to misfolded proteins and fixes the misfolded state. This review provides an overview of the mechanism and consequences of oxidative proteome damage - specifically irreversible protein carbonylation - in relation to ageing, including that of the skin as well as to age-related degeneration and diseases (ARDD) and their mitigation. A literature review of published manuscripts, available from PubMed, focusing on proteome, proteostasis, proteotoxicity, protein carbonylation, related inflammatory diseases, ARDD and the impact of the damaged proteome on ageing. During ageing, proteome damage, especially protein carbonylation, correlates with biological age. Carbonylated proteins form aggregates which can be considered as markers and accelerators of ageing and are common markers of most ARDD. Protein carbonylation leads to general ageing of the organism and organs including the skin and potentially to diseases including Alzheimer and Parkinson disease, diabetes, psoriasis, and skin cancer. Current research is promising and may open new therapeutic approaches and perspectives by targeting proteome protection as an age and ARDD management strategy.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Enhancing saccharification of wheat straw by mixing enzymes from genetically-modified Trichoderma reesei and Aspergillus niger.

OBJECTIVES: To increase the efficiency of enzymatic hydrolysis for plant biomass conversion into renewable biofuel and chemicals. RESULTS: By overexpressing the point mutation A824 V transcriptional activator Xyr1 in Trichoderma reesei, carboxymethyl cellulase, cellobiosidase and ß-D-glucosidase activities of the best mutant were increased from 1.8 IU/ml, 0.1 IU/ml and 0.05 IU/ml to 4.8 IU/ml, 0.4 IU/ml and 0.3 IU/ml, respectively. The sugar yield of wheat straw saccharification by combining enzymes from this mutant and the Aspergillus niger genetically modified strain ΔcreA/xlnR c/araR c was improved up to 7.5 mg/ml, a 229 % increase compared to the combination of wild type strains. CONCLUSIONS: Mixing enzymes from T. reesei and A. niger combined with the genetic modification of transcription factors is a promising strategy to increase saccharification efficiency.

Spatial differentiation of gene expression in Aspergillus niger colony grown for sugar beet pulp utilization.

Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

BACKGROUND: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. RESULTS: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. CONCLUSIONS: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.

Aromatic metabolism of filamentous fungi in relation to the presence of aromatic compounds in plant biomass.

The biological conversion of plant lignocellulose plays an essential role not only in carbon cycling in terrestrial ecosystems but also is an important part of the production of second generation biofuels and biochemicals. The presence of the recalcitrant aromatic polymer lignin is one of the major obstacles in the biofuel/biochemical production process and therefore microbial degradation of lignin is receiving a great deal of attention. Fungi are the main degraders of plant biomass, and in particular the basidiomycete white rot fungi are of major importance in converting plant aromatics due to their ability to degrade lignin. However, the aromatic monomers that are released from lignin and other aromatic compounds of plant biomass are toxic for most fungi already at low levels, and therefore conversion of these compounds to less toxic metabolites is essential for fungi. Although the release of aromatic compounds from plant biomass by fungi has been studied extensively, relatively little attention has been given to the metabolic pathways that convert the resulting aromatic monomers. In this review we provide an overview of the aromatic components of plant biomass, and their release and conversion by fungi. Finally, we will summarize the applications of fungal systems related to plant aromatics.

Sugar catabolism in Aspergillus and other fungi related to the utilization of plant biomass.

Fungi are found in all natural and artificial biotopes and can use highly diverse carbon sources. They play a major role in the global carbon cycle by decomposing plant biomass and this biomass is the main carbon source for many fungi. Plant biomass is composed of cell wall polysaccharides (cellulose, hemicellulose, pectin) and lignin. To degrade cell wall polysaccharides to different monosaccharides, fungi produce a broad range of enzymes with a large variety in activities. Through a series of enzymatic reactions, sugar-specific and central metabolic pathways convert these monosaccharides into energy or metabolic precursors needed for the biosynthesis of biomolecules. This chapter describes the carbon catabolic pathways that are required to efficiently use plant biomass as a carbon source. It will give an overview of the known metabolic pathways in fungi, their interconnections, and the differences between fungal species.

Bacillus subtilis attachment to Aspergillus niger hyphae results in mutually altered metabolism.

Interaction between microbes affects the growth, metabolism and differentiation of members of the microbial community. While direct and indirect competition, like antagonism and nutrient consumption have a negative effect on the interacting members of the population, microbes have also evolved in nature not only to fight, but in some cases to adapt to or support each other, while increasing the fitness of the community. The presence of bacteria and fungi in soil results in various interactions including mutualism. Bacilli attach to the plant root and form complex communities in the rhizosphere. Bacillus subtilis, when grown in the presence of Aspergillus niger, interacts similarly with the fungus, by attaching and growing on the hyphae. Based on data obtained in a dual transcriptome experiment, we suggest that both fungi and bacteria alter their metabolism during this interaction. Interestingly, the transcription of genes related to the antifungal and putative antibacterial defence mechanism of B. subtilis and A. niger, respectively, are decreased upon attachment of bacteria to the mycelia. Analysis of the culture supernatant suggests that surfactin production by B. subtilis was reduced when the bacterium was co-cultivated with the fungus. Our experiments provide new insights into the interaction between a bacterium and a fungus.

A genomic survey of proteases in Aspergilli.

BACKGROUND: Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide range of biochemical properties. Several Aspergilli have the ability to produce a variety of proteases, but no comprehensive comparative study has been carried out on protease productivity in this genus so far. RESULTS: We have performed a combined analysis of comparative genomics, proteomics and enzymology tests on seven Aspergillus species grown on wheat bran and sugar beet pulp. Putative proteases were identified by homology search and Pfam domains. These genes were then clusters based on orthology and extracellular proteases were identified by protein subcellular localization prediction. Proteomics was used to identify the secreted enzymes in the cultures, while protease essays with and without inhibitors were performed to determine the overall protease activity per protease class. All this data was then integrated to compare the protease productivities in Aspergilli. CONCLUSIONS: Genomes of Aspergillus species contain a similar proportion of protease encoding genes. According to comparative genomics, proteomics and enzymatic experiments serine proteases make up the largest group in the protease spectrum across the species. In general wheat bran gives higher induction of proteases than sugar beet pulp. Interesting differences of protease activity, extracellular enzyme spectrum composition, protein occurrence and abundance were identified for species. By combining in silico and wet-lab experiments, we present the intriguing variety of protease productivity in Aspergilli.

The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown.

BACKGROUND: Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. RESULTS: The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. CONCLUSIONS: With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.

Regulation of plant biomass utilization in Aspergillus.

The ability of fungi to survive in every known biotope, both natural and man-made, relies in part on their ability to use a wide range of carbon sources. Fungi degrade polymeric carbon sources present in the environment (polysaccharides, proteins, and lignins) to use the monomeric components as nutrients. However, the available carbon sources vary strongly in nature, both between biotopes and in time. The degradation of polymeric carbon sources is mediated through the production of a broad range of enzymes, the production of which is tightly controlled by a network of regulators and linked to the activation of catabolic pathways to convert the released monomers. This review summarizes the knowledge of Aspergillus regulators involved in plant biomass utilization.

Nutritional physiology of a rock-inhabiting, model microcolonial fungus from an ancestral lineage of the Chaetothyriales (Ascomycetes).

Rock-inhabiting black fungi [also microcolonial or meristematic fungi (MCF)] are a phylogenetically diverse group of melanised ascomycetes with distinctive morphological features that confer extensive stress tolerance and permit survival in hostile environments. The MCF strain A95 Knufia petricola (syn. Sarcinomyces petricola) belongs to an ancestral lineage of the order Chaetothyriales (class Eurotiomycetes). K. petricola strain A95 is a rock-inhabiting MCF and its growth requirements were studied using the 96-well plate-based Biolog System under ∼1070 different conditions (osmotic stress, pH growth optima, growth factor requirements and nutrient catabolism). A95 is an osmotolerant, oligotrophic MCF that grows best around pH 5. Remarkably, A95 shows metabolic activity in the absence of added nitrogen, phosphorus or sulphur. Correlations could be drawn between the known nutrient requirements of A95 and what probably is available in sub-aerial systems (rock and other material surfaces). Detailed knowledge of A95's metabolic requirements allowed formulation of a synthetic medium that supports strong fungal growth.

Degradation of different pectins by fungi: correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin.

BACKGROUND: Pectins are diverse and very complex biomolecules and their structure depends on the plant species and tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins have positive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes are required. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range of pectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis. RESULTS: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that the presence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degrade pectins. However, this correlation is less clear when we zoom in to the pectic structural elements. CONCLUSIONS: This study highlights the complexity of the mechanisms involved in fungal degradation of complex carbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards the production of specific pectinolytic fractions.

The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes.

Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Serum resistance to singlet oxygen in patients with diabetes mellitus in comparison to healthy donors.

Diabetes mellitus causes endothelial injury through oxidative stress involving reactive oxygen species and peroxides as well as inflammation, both of which consume antioxidant defenses. Singlet oxygen ((1)O(2)) is produced by leukocytes during inflammatory and biochemical reactions and deactivated by producing reactive oxygen species and peroxides. To determine whether serum was capable of deactivating (1)O(2), we triggered a photo reaction in sera from 53 healthy donors and 52 diabetic patients. Immediately after light delivery, dichlorofluorescein was added and then its fluorescence was recorded. The mean capacity of (1)O(2) or secondary oxidant deactivation was reduced in patients with diabetes mellitus. Hemolysis reduced deactivation of (1)O(2)-induced secondary oxidants in both healthy and diabetic patients. Body mass index, age, platelet counts, and blood cell numbers exerted a nonlinear influence. High levels of glycated hemoglobin were associated with an increased deactivation of oxidative species, whereas high-density lipoprotein cholesterol, total cholesterol, and the total cholesterol to high-density lipoprotein cholesterol ratio decreased the serum deactivation capacity. Oral antidiabetics bore no influence on deactivation, which was restored by insulin in women. Deactivation capacity was lower in women, who had half the complications found in men, suggesting that, with more severe diabetes mellitus, protection was maintained against complications. Resistance to (1)O(2) should be considered during the monitoring of diabetes mellitus.

Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level.

BACKGROUND: Rhizopus oryzae is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus, causing Rhizopus rot and mucomycoses. RESULTS: Carbohydrate Active enzyme (CAZy) annotation of the R. oryzae identified, in contrast to other filamentous fungi, a low number of glycoside hydrolases (GHs) and a high number of glycosyl transferases (GTs) and carbohydrate esterases (CEs). A detailed analysis of CAZy families, supported by growth data, demonstrates highly specialized plant and fungal cell wall degrading abilities distinct from ascomycetes and basidiomycetes. The specific genomic and growth features for degradation of easily digestible plant cell wall mono- and polysaccharides (starch, galactomannan, unbranched pectin, hexose sugars), chitin, chitosan, ß-1,3-glucan and fungal cell wall fractions suggest specific adaptations of R. oryzae to its environment. CONCLUSIONS: CAZy analyses of the genome of the zygomycete fungus R. oryzae and comparison to ascomycetes and basidiomycete species revealed how evolution has shaped its genetic content with respect to carbohydrate degradation, after divergence from the Ascomycota and Basidiomycota.

Post-genomic approaches to understanding interactions between fungi and their environment.

Fungi inhabit every natural and anthropogenic environment on Earth. They have highly varied life-styles including saprobes (using only dead biomass as a nutrient source), pathogens (feeding on living biomass), and symbionts (co-existing with other organisms). These distinctions are not absolute as many species employ several life styles (e.g. saprobe and opportunistic pathogen, saprobe and mycorrhiza). To efficiently survive in these different and often changing environments, fungi need to be able to modify their physiology and in some cases will even modify their local environment. Understanding the interaction between fungi and their environments has been a topic of study for many decades. However, recently these studies have reached a new dimension. The availability of fungal genomes and development of post-genomic technologies for fungi, such as transcriptomics, proteomics and metabolomics, have enabled more detailed studies into this topic resulting in new insights. Based on a Special Interest Group session held during IMC9, this paper provides examples of the recent advances in using (post-)genomic approaches to better understand fungal interactions with their environments.

PCSK9 dominant negative mutant results in increased LDL catabolic rate and familial hypobetalipoproteinemia.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central player in the regulation of cholesterol homeostasis, increasing the low-density lipoprotein (LDL) receptor degradation. Our study aimed at exploring the pathogenic consequences in vivo and in vitro of a PCSK9 prodomain mutation found in a family with hypobetalipoproteinemia (FHBL). METHODS AND RESULTS: A white 49-year-old diabetic man had profound FBHL (LDLC: 16 mg/dL) whereas his daughter and sister displayed a milder phenotype (LDLC 44 mg/dL and 57 mg/dL, respectively), all otherwise healthy with a normal liver function. A monoallelic PCSK9 double-mutant R104C/V114A cosegregated with FBHL, with no mutation found at other FHBL-causing loci. A dose-effect was also found in FBHL relatives for plasma APOB and PCSK9 (very-low to undetectable in proband, approximately 50% decreased in sister and daughter) and LDL catabolic rate (256% and 88% increased in proband and daughter). Transient transfection in hepatocytes showed severely impaired processing and secretion of the double mutant which acted as a dominant negative over secretion of wild-type PCSK9. CONCLUSIONS: These results show that heterozygous PCSK9 missense mutations may associate with profound hypobetalipoproteinemia and constitute the first direct evidence in human that decrease of plasma LDLC concentrations associated to PCSK9 LOF mutations are attributable to an increased clearance rate of LDL.