Vivian de los Ríos

Aspergillus RabBRab5 Integrates Acquisition of Degradative Identity with the Long Distance Movement of Early Endosomes

Of the two Aspergillus early endosomal Rab5 paralogues, RabB recruits, in its GTP conformation, Vps19, Vps45, and Vps34, and the CORVET complex and couples acquisition of PI(3)P degradative identity with the long-distance movement of early endosomes. RabA also recruits CORVET, albeit less efficiently. The simultaneous loss of RabA and RabB is lethal.

Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment.

BackgroundIdentifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases.ResultsIn wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases.ConclusionThe protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis.

Protein haptenation by amoxicillin: High resolution mass spectrometry analysis and identification of target proteins in serum

Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including β-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.

Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein- mediated basipetal movement

Highly motile fungal early endosomes can be easily distinguished from more static late endosomes and vacuoles, a feature that is exploited to study endosomal maturation. RabA/RabB early endosomes mature into RabSRab7 late endosomes as they move away from the tip where endocytosis predominates, augmenting their size, with concomitant loss of motility.

TRAPPII regulates exocytic Golgi exit by mediating nucleotide exchange on the Ypt31 ortholog RabERAB11

Significance Ypt1 and Ypt31/32 RAB GTPases regulate traffic across the Golgi. Both are activated by TRAPP, an oligomeric GEF. Three TRAPP versions share the same core subunits. TRAPPI and TRAPPIII activate Ypt1. The third, TRAPPII, composed of TRAPPI plus specific subunits, appears to act specifically on Ypt31, but this role has been disputed. By combining the resolving power of fungal genetics with biochemical assays, we establish that the physiological target of TRAPPII is RabE, the Aspergillus Ypt31 ortholog. However, our data suggest that TRAPPII contains independent binding sites for RabE and RabO (Ypt1), possibly explaining its relative lack of discrimination in vitro. TRAPPII arrives at Golgi cisternae preceding their dissipation into carriers, determining the Golgi–to–post-Golgi transition through RabE recruitment. The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabORAB1 resides in the Golgi, RabERAB11 localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabERAB11, but not RabORAB1, immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabERAB11 in vivo. hypA1ts rapidly shifts RabERAB11, but not RabORAB1, to the cytosol, consistent with HypATrs120 being specifically required for RabERAB11 activation. Missense mutations rescuing hypA1ts at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabORAB1 did not, establishing that the essential role of TRAPPII is facilitating RabERAB11 nucleotide exchange. In vitro, TRAPPII purified with HypATrs120-S-tag accelerates nucleotide exchange on RabERAB11 and, paradoxically, to a lesser yet substantial extent, on RabORAB1. Evidence obtained by exploiting hypA1-mediated destabilization of HypATrs120/HypCTrs130/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabORAB1, which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypATrs120 colocalizes with RabERAB11, arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi–to–post-Golgi transition.

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (lf-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP *) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. Recombinant AFP * produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will permit investigation of the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches.

Proteomic analysis of 1α,25-Dihydroxyvitamin D3 action on human colon cancer cells reveals a link to splicing regulation

1α,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and other vitamin D compounds are promising molecules in the prevention and therapy of colon cancer and other neoplasias. To study the mechanism of action of 1,25(OH)(2)D(3) in colon cancer cells, we carried out a comparative proteomic analysis of the nuclear fractions of SW480-ADH cells treated with 1,25(OH)(2)D(3) or vehicle during 8 or 48h. 2D-DIGE analysis combined with MALDI-TOF-TOF mass spectrometry interrogation led to the identification of 59 differentially expressed unique proteins. Most identified proteins were nuclear, but several cytoskeleton-associated proteins were also detected. A good concordance between changes in expression at protein and RNA levels was observed for the validated proteins. A large group of identified proteins, such as SFPQ, SMARCE, KHSRP, TARDBP and PARP1, were involved in RNA processing or modification and have been ascribed to the spliceosome compartment of human cells. In addition, a smaller group of proteins (ERM (Ezrin, Radixin, Moesin) family, VCL, CORO1C, ACTB) were cytoskeleton-associated and played a role in cell adhesion and morphology. These results confirm the induction of epithelial phenotype by 1,25(OH)(2)D(3) and suggest a role for vitamin D compounds in the regulation of the spliceosome and thus, in alternative splicing and possibly microRNA synthesis in colon cancer cells.

Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr181, whereas H2O2-LiP contains a hydroxylated Tyr181. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr181 and Phe89. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine-VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine-VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.

GmcA Is a Putative Glucose-Methanol-Choline Oxidoreductase Required for the Induction of Asexual Development in Aspergillus nidulans

Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ΔflbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ΔgmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ΔgmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation.

Endocytic recycling via the TGN underlies the polarized hyphal mode of life

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.