Identification of Protein Complexes from Filamentous Fungi with Tandem Affinity Purification

Bayram, Özgür; Valerius, Oliver; Jöhnk, Bastian; Braus, Gerhard H.

doi:10.1007/978-1-62703-122-6_14

Cited by 30 publications

(32 citation statements)

References 19 publications

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“…Affinity purification was performed as described in reference 71. Eight 1,000-ml Erlenmeyer flasks each containing 200 ml of GX-MM were inoculated with 5 × 10 6 conidia per ml.…”

Section: Methodsmentioning

confidence: 99%

A Class 1 Histone Deacetylase with Potential as an Antifungal Target

Bauer

Varadarajan

Pidroni

et al. 2016

mBio

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Histone deacetylases (HDACs) remove acetyl moieties from lysine residues at histone tails and nuclear regulatory proteins and thus significantly impact chromatin remodeling and transcriptional regulation in eukaryotes. In recent years, HDACs of filamentous fungi were found to be decisive regulators of genes involved in pathogenicity and the production of important fungal metabolites such as antibiotics and toxins. Here we present proof that one of these enzymes, the class 1 type HDAC RpdA, is of vital importance for the opportunistic human pathogen Aspergillus fumigatus. Recombinant expression of inactivated RpdA shows that loss of catalytic activity is responsible for the lethal phenotype of Aspergillus RpdA null mutants. Furthermore, we demonstrate that a fungus-specific C-terminal region of only a few acidic amino acids is required for both the nuclear localization and catalytic activity of the enzyme in the model organism Aspergillus nidulans. Since strains with single or multiple deletions of other classical HDACs revealed no or only moderate growth deficiencies, it is highly probable that the significant delay of germination and the growth defects observed in strains growing under the HDAC inhibitor trichostatin A are caused primarily by inhibition of catalytic RpdA activity. Indeed, even at low nanomolar concentrations of the inhibitor, the catalytic activity of purified RpdA is considerably diminished. Considering these results, RpdA with its fungus-specific motif represents a promising target for novel HDAC inhibitors that, in addition to their increasing impact as anticancer drugs, might gain in importance as antifungals against life-threatening invasive infections, apart from or in combination with classical antifungal therapy regimes.

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“…Affinity purification was performed as described in reference 71. Eight 1,000-ml Erlenmeyer flasks each containing 200 ml of GX-MM were inoculated with 5 × 10 6 conidia per ml.…”

Section: Methodsmentioning

confidence: 99%

A Class 1 Histone Deacetylase with Potential as an Antifungal Target

Bauer

Varadarajan

Pidroni

et al. 2016

mBio

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“…The tagged melanin proteins were all expressed using their native promoters. The protein extraction procedure was the same as previously described (50, 51). Aliquots of proteins were separated on SDS gels for Western blot analysis using the anti-FLAG (catalog number F2426; Sigma) or the anti-GFP (catalog number 11814460001; Roche) monoclonal antibody.…”

Section: Methodsmentioning

confidence: 99%

“…Conidia harvested from colonies cultured on MM solid medium for 2 days were used for protein extraction. Total proteins were extracted using the previously described method (50, 51)…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, the total proteins extracted from young conidia were processed by following a sequence of three in vitro chemical steps and one step of purification of the originally palmitoylated proteins with streptavidin, as follows. (i) The total proteins were extracted from conidia homogenized with a bead beater or mortar and pestle as described previously (50, 51). The proteins were resuspended in lysis buffer containing N -ethylmaleimide (NEM) (catalog number PI 23030; Pierce) overnight, which blocks the free thiols of the native proteins.…”

Section: Methodsmentioning

confidence: 99%

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Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

Upadhyay

Lin

2016

mBio

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Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

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“…To perform reciprocal Co-IP assays, C-terminal HA-tagged SdsA strain was used. Briefly, mycelia were frozen with liquid nitrogen and ground, and 500 mg was resuspended in 1 ml of B250 buffer (Bayram et al, 2012). Samples were centrifuged at maximum speed for 10 min at 4°C.…”

Section: Construction Of the A Fumigatus Mutantsmentioning

confidence: 99%

Aspergillus fumigatusprotein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence

Manfiolli

Castro

Reis

et al. 2017

Cellular Microbiology

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Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.

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Identification of Protein Complexes from Filamentous Fungi with Tandem Affinity Purification

Cited by 30 publications

References 19 publications

A Class 1 Histone Deacetylase with Potential as an Antifungal Target

A Class 1 Histone Deacetylase with Potential as an Antifungal Target

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

Aspergillus fumigatusprotein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence

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