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Miguel A Peñalva

Publications and source records attributed to Miguel A Peñalva.

14 recordsLinked to original sources
PubMed2005

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Allergens↗
PubMed2005

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae.

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Aspergillus fumigatus↗
PubMed2005

Tracing the endocytic pathway of Aspergillus nidulans with FM4-64.

Simple procedures using FM4-64 to follow membrane internalization and transport to the vacuolar system and endomembranes in Aspergillus nidulans are described. FM4-64 internalization is energy, temperature and F-actin dependent, strongly suggesting that it occurs by endocytosis. The dye sequentially labels: (i) cortical punctuate organelles whose motility resembles that of yeast actin patches; (ii) approximately 0.7 microm circular, hollow structures representing mature endosome/vacuole; and (iii) intermediate and large (2-3 microm in diameter) size vacuoles whose lumen is strongly labeled with 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). These large vacuoles possibly correspond to the final stage of one branch of the endocytic pathway. In addition, FM4-64 labels strongly the mitochondrial network and weakly the nuclear membrane. A class of cytoplasmic punctuate organelles which become fluorescent very shortly after dye loading and that can move in either apical or basal direction at an average rate of 2-3 microm s(-1) is also described. This work provides a useful framework for the phenotypic characterization of A. nidulans mutants affected in endocytosis.

Aspergillus nidulans↗
PubMed2005

Arrestin-related proteins mediate pH signaling in fungi.

Metazoan arrestins bind to seven-transmembrane (7TM) receptors to regulate function. Aspergillus nidulans PalF, a protein involved in the fungal ambient pH signaling pathway, contains arrestin N-terminal and C-terminal domains and binds strongly to two different regions within the C-terminal cytoplasmic tail of the 7TM, putative pH sensor PalH. Upon exposure to alkaline ambient pH, PalF is phosphorylated and, like mammalian beta-arrestins, ubiquitinated in a signal-dependent and 7TM protein-dependent manner. Substitution in PalF of a highly conserved arrestin N-terminal domain Ser residue prevents PalF-PalH interaction and pH signaling in vivo. Thus, PalF is the first experimentally documented fungal arrestin-related protein, dispelling the notion that arrestins are restricted to animal proteomes. Epistasis analyses demonstrate that PalF posttranslational modification is partially dependent on the 4TM protein PalI but independent of the remaining pH signal transduction pathway proteins PalA, PalB, and PalC, yielding experimental evidence bearing on the order of participation of the six components of the pH signal transduction pathway. Our data strongly implicate PalH as an ambient pH sensor, possibly with the cooperation of PalI.

Amino Acid Sequence↗
PubMed2005

Mutational analysis of the pH signal transduction component PalC of Aspergillus nidulans supports distant similarity to BRO1 domain family members.

The alkaline ambient pH signal transduction pathway component PalC has no assigned molecular role. Therefore we attempted a gene-specific mutational analysis and obtained 55 new palC loss-of-function alleles including 24 single residue substitutions. Refined similarity searches reveal conserved PalC regions including one with convincing similarity to the BRO1 domain, denoted PCBROH, where clustering of mutational changes, including PCBROH key residue substitutions, supports its structural and/or functional importance. Since the BRO1 domain occurs in the multivesicular body (MVB) pathway protein Bro1/Vps31 and also the pH signal transduction protein PalA (Rim20), both of which interact with MVB component (ESCRT-III protein) Vps32/Snf7, this might reflect a further link between the pH response and endocytosis.

Amino Acid Sequence↗
PubMed2004

Fungal metabolic model for type I 3-methylglutaconic aciduria.

Aspergillus nidulans catabolizes Leu to acetyl-CoA and acetoacetate through a pathway homologous to that used by humans. Fungal hlyA encodes a bifunctional polypeptide comprising the last two enzymes in this pathway, 3-methylglutaconyl-CoA hydratase and 3-hydroxy-3-methylglutaryl-CoA lyase. hlyA transcription is specifically induced by Leu. A Delta hlyA mutation removing the complete 3-methylglutaconyl-CoA hydratase C-terminal domain prevents growth on Leu but not on lactose or other amino acids and, in agreement with the predicted enzyme function, leads to Leu-dependent accumulation of 3-methylglutaconic acid in the culture supernatant. These data represent a formal demonstration in vivo of the specific involvement of 3-methylglutaconyl-CoA hydratase in Leu catabolism. Type I 3-methylglutaconic aciduria patients deficient in 3-methylglutaconyl-CoA hydratase show urinary excretion of 3-methylglutaconic acid and, in contrast to the other three types of methylglutaconic acidurias, 3-hydroxyisovaleric acid excretion. Gas chromatography-mass spectrometry analysis revealed an accumulation of both diagnostic compounds in Delta hlyA culture supernatants, illustrating that the metabolic consequences of equivalent inborn errors of metabolism are conserved from fungi to humans. Using our fungal type I 3-methylglutaconic aciduria model, we show that metabolites accumulating in the deficient strain are toxic, although less so than those accumulating in a Delta mccB strain deficient for the upstream enzyme 3-methylcrotonyl-CoA carboxylase. Diagnostic metabolite accumulation is Leu concentration-dependent, in agreement with the ability of Leu intake restriction to reduce the levels of offending metabolites. Delta mccB and Delta hlyA mutations show additive Leu toxicities. The double mutant accumulates 3-methylglutaconic acid, which can therefore be synthesized through 3-methylcrotonyl-CoA carboxylase-dependent and -independent reactions.

Alleles↗
PubMed2004

Recent advances in the characterization of ambient pH regulation of gene expression in filamentous fungi and yeasts.

All microorganisms must adapt to the pH of their environment. One aspect of this adaptation, particularly important for organisms that grow over a wide pH range, is the ability to express appropriately genes whose roles ultimately involve functions at the cell surface or in the environment. Genes encoding permeases, secreted enzymes, enzymes involved in synthesis of exported metabolites such as toxins and antibiotics, and probably enzymes modifying secreted proteins posttranslationally all fall into this category. Here we discuss the most recent findings on the transcriptional regulatory system in fungi that enables such genes to be expressed only when the ambient pH is conducive to their ultimate functions. The intriguing issue of how pH is sensed and how the resulting signal is transmitted to the transcription factor involves at least one late endosome component. Proper functioning of the regulatory system responding to ambient pH is essential for fungal pathogenicity of both animals and plants.

Aspergillus nidulans↗
PubMed2003

Overlap of nuclear localisation signal and specific DNA-binding residues within the zinc finger domain of PacC.

The transcription factor PacC, mediating regulation of gene expression by ambient pH in the genetically amenable fungus Aspergillus nidulans, contains a three zinc finger DNA-binding domain (zf-DBD) including a nuclear localisation signal (NLS). We selected 38 novel mutations impairing PacC function, of which 21 missense mutations identify individual residues essential for zf-DBD structure/function. Our functional analysis agrees with our previous conclusion that finger 1 does not bind DNA and provides in vivo evidence that Trp80 and Trp116, located in the Cys knuckles of adjacent zinc fingers, are critical for zf-DBD structure/function. In the finger 3 alpha-helix, Gln155 (+4) is specifically involved in contacting DNA, while the major role of Lys159 (+6) resides in the nuclear localisation of the protein. In contrast, Lys158 is essential for DNA binding and for nuclear localisation. As finger 3 suffices to drive nuclear localisation of green fluorescent protein, we conclude that it contains an NLS including essential Lys158 and Lys159. These residues are within an alpha-helical basic sequence that is completely conserved amongst zinc fingers of the PacC/RIM101 family and present in an identical position of the last finger alpha-helix of Drosophila Cubitus interruptus, where it is also involved in nuclear localisation. We propose that PacC and Gli/Ci zf-DBDs belong to a subclass of these domains characterised by possession of a pair of conserved Trp residues involved in the interaction between the two most N-terminal fingers and the presence of an NLS in the alpha-helix of the most C-terminal finger. Loss of PacC nuclear localisation resulting from His142Leu (beta-strand) and Phe151Ser (hydrophobic core) substitutions in finger 3 suggests that its folding is required for NLS function. Overlap of DNA binding and NLS may aid release of PacC from its cognate importer(s) upon nuclear translocation, as suggested for zinc binuclear cluster proteins.

Active Transport, Cell Nucleus↗
PubMed2003

Fungal metabolic model for 3-methylcrotonyl-CoA carboxylase deficiency.

Aspergillus nidulans is able to use Leu as the sole carbon source through a metabolic pathway leading to acetyl-CoA and acetoacetate that is homologous to that used by humans. mccA and mccB, the genes encoding the subunits of 3-methylcrotonyl-CoA carboxylase, are clustered with ivdA encoding isovaleryl-CoA dehydrogenase, a third gene of the Leu catabolic pathway, on the left arm of chromosome III. Their transcription is induced by Leu and other hydrophobic amino acids and repressed by glucose. Phenotypically indistinguishable DeltamccA, DeltamccB, and DeltamccA DeltamccB mutations prevent growth on Leu but not on lactose or other amino acids, formally demonstrating in vivo the specific involvement of 3-methylcrotonyl-CoA carboxylase in Leu catabolism. Growth of mcc mutants on lactose plus Leu is impaired, indicating that Leu metabolite(s) accumulation resulting from the metabolic block is toxic. Human patients carrying loss-of-function mutations in the genes encoding the subunits of 3-methylcrotonyl-CoA carboxylase suffer from methylcrotonylglycinuria. Gas chromatography/mass spectrometry analysis of culture supernatants revealed that fungal Deltamcc strains accumulate 3-hydroxyisovaleric acid, one of the diagnostic compounds in the urine of these patients, illustrating the remarkably similar consequences of equivalent genetic errors of metabolism in fungi and humans. We use our fungal model(s) for methylcrotonylglycinuria to show accumulation of 3-hydroxyisovalerate on transfer of 3-methylcrotonyl-CoA carboxylase-deficient strains to the isoprenoid precursors acetate, 3-hydroxy-3-methylglutarate, or mevalonate. This represents the first reported genetic evidence for the existence of a metabolic link involving 3-methylcrotonyl-CoA carboxylase between isoprenoid biosynthesis and Leu catabolism, providing additional support to the mevalonate shunt proposed previously (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71).

Alleles↗
PubMed2003

pH regulation in Aspergillus and parallels with higher eukaryotic regulatory systems.

All organisms respond to their environment to some extent, and for many microbes the variation in environment can be enormous. An important asset for coping with environmental variation is physiological versatility--a hallmark of many fungi. The ability of fungi to thrive over a wide range of pH is partly due to a genetic regulatory system that tailors gene expression to the ambient pH. Here we focus on the pH regulatory system of Aspergillus nidulans, where a novel signal transduction (pal) pathway mediates the first of two steps in the proteolytic processing of a transcription factor (PacC). Such processing is reminiscent of that of some well-known higher eukaryotic transcription factors, such as Cubitus interruptus, NF-kappa B and sterol regulatory element binding proteins. Intriguingly, endocytosis seems to be connected to pH signalling.

Amino Acid Sequence↗
PubMed2003

YPXL/I is a protein interaction motif recognized by aspergillus PalA and its human homologue, AIP1/Alix.

The zinc finger transcription factor PacC undergoes two-step proteolytic activation in response to alkaline ambient pH. PalA is a component of the fungal ambient pH signal transduction pathway. Its mammalian homologue AIP1/Alix interacts with the apoptosis-linked protein ALG-2. We show that both PalA and AIP1/Alix recognize a protein-protein binding motif that we denote YPXL/I, where Tyr, Pro, and Leu/Ile are crucial for its interactive properties. Two such motifs flanking the signaling protease cleavage site mediate direct binding of PalA to PacC, required for the first and only pH-regulated cleavage of this transcription factor. PalA can bind the "closed" (i.e., wild-type full-length) conformer of PacC, suggesting that PalA binding constitutes the first stage in the two-step proteolytic cascade, recruiting or facilitating access of the signaling protease, presumably PalB. In addition to recognizing YPXL/I motifs, both PalA and AIP1/Alix interact with the Aspergillus class E Vps protein Vps32 homologue, a member of a protein complex involved in the early steps of the multivesicular body pathway, suggesting that this interaction is an additional feature of proteins of the PalA/AIP1/Alix family.

Alleles↗
PubMed2002

Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps.

The Aspergillus PacC transcription factor undergoes proteolytic activation in response to alkaline ambient pH. In acidic environments, the 674 residue translation product adopts a 'closed' conformation, protected from activation through intramolecular interactions involving the < or = 150 residue C-terminal domain. pH signalling converts PacC to an accessible conformation enabling processing cleavage within residues 252--254. We demonstrate that activation of PacC requires two sequential proteolytic steps. First, the 'closed' translation product is converted to an accessible, committed intermediate by proteolytic elimination of the C-terminus. This ambient pH-regulated cleavage is required for the final, pH-independent processing reaction and is mediated by a distinct signalling protease (possibly PalB). The signalling protease cleaves PacC between residues 493 and 500, within a conserved 24 residue 'signalling protease box'. Precise deletion or Leu498Ser substitution prevents formation of the committed and processed forms, demonstrating that signalling cleavage is essential for final processing. In contrast, signalling cleavage is not required for processing of the Leu340Ser protein, which lacks interactions preventing processing. In its two-step mechanism, PacC processing can be compared with regulated intramembrane proteolysis.

Amino Acid Sequence↗
PubMed2002

Regulation of gene expression by ambient pH in filamentous fungi and yeasts.

Life, as we know it, is water based. Exposure to hydroxonium and hydroxide ions is constant and ubiquitous, and the evolutionary pressure to respond appropriately to these ions is likely to be intense. Fungi respond to their environments by tailoring their output of activities destined for the cell surface or beyond to the ambient pH. We are beginning to glimpse how they sense ambient pH and transmit this information to the transcription factor, whose roles ensure that a suitable collection of gene products will be made. Although relatively little is known about pH signal transduction itself, its consequences for the cognate transcription factor are much clearer. Intriguingly, homologues of components of this system mediating the regulation of fungal gene expression by ambient pH are to be found in the animal kingdom. The potential applied importance of this regulatory system lies in its key role in fungal pathogenicity of animals and plants and in its control of fungal production of toxins, antibiotics, and secreted enzymes.

Animal Diseases↗