scholarly journals An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

2013 ◽  
Vol 12 (6) ◽  
pp. 816-827 ◽  
Author(s):  
Silke Palzer ◽  
Yannick Bantel ◽  
Franziska Kazenwadel ◽  
Michael Berg ◽  
Steffen Rupp ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Code ◽  
Molecular Interactions ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Content Type ◽  
Suppressor Trna

ABSTRACT For novel insights into the pathogenicity of Candida albicans , studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins in vivo are scarce, we expanded the genetic code of C. albicans with the synthetic photo-cross-linking amino acid p -azido- l -phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyl-tRNA synthetase and a suppressor tRNA pair ( Ec TyrtRNA CUA ) derived from Escherichia coli , which was previously reported to be orthogonal in Saccharomyces cerevisiae . We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and Ec TyrtRNA CUA for C. albicans and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecular-weight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of C. albicans with the unnatural photo-cross-linker amino acid AzF and applying it for in vivo binary protein interaction analyses is feasible.

scholarly journals The Paralogous Transcription Factors Stp1 and Stp2 of Candida albicans Have Distinct Functions in Nutrient Acquisition and Host Interaction

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Pedro Miramón ◽  
Andrew W. Pountain ◽  
Ambro van Hoof ◽  
Michael C. Lorenz
Keyword(s):  
Candida Albicans ◽  
Transcription Factors ◽  
Amino Acid ◽  
Cell Damage ◽  
Nutrient Acquisition ◽  
Acid Uptake ◽  
Content Type ◽  
Host Interaction ◽  
Amino Acid Utilization

ABSTRACT Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1Δ/Δ and stp2Δ/Δ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1Δ/Δ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.

scholarly journals The eucaryotic aminoacyl-tRNA synthetase complex: suggestions for its structure and function.

1984 ◽  
Vol 99 (2) ◽  
pp. 373-377 ◽  
Author(s):  
M P Deutscher
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Structural Similarity ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Membrane Bound ◽  
And Function ◽  
Molecular Weight Form

Aminoacyl-tRNA synthetases from eucaryotic cells generally are isolated as high molecular weight complexes comprised of multiple synthetase activities, and often containing other components as well. A model is proposed for the synthetase complex in which hydrophobic extensions on the proteins serve to maintain them in their high molecular weight form, but are not needed for catalytic activity. The structural similarity of these enzymes to certain membrane-bound proteins, and its implications for synthetase localization and function in vivo, are discussed.

scholarly journals Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

2018 ◽  
Author(s):  
Birthe Meineke ◽  
Johannes Heimgärtner ◽  
Lorenzo Lafranchi ◽  
Simon J Elsässer
Keyword(s):  
Genetic Code ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Human Gut ◽  
Methanosarcina Mazei ◽  
Genetic Code Expansion

ABSTRACTGenetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

scholarly journals Evolution of Life on Earth: tRNA, Aminoacyl-tRNA Synthetases and the Genetic Code

Life ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 21 ◽  
Author(s):  
Lei Lei ◽  
Zachary F Burton
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Elongation Factor ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Amino Acid Code ◽  
Evolution Of Life ◽  
Life On Earth

Life on Earth and the genetic code evolved around tRNA and the tRNA anticodon. We posit that the genetic code initially evolved to synthesize polyglycine as a cross-linking agent to stabilize protocells. We posit that the initial amino acids to enter the code occupied larger sectors of the code that were then invaded by incoming amino acids. Displacements of amino acids follow selection rules. The code sectored from a glycine code to a four amino acid code to an eight amino acid code to an ~16 amino acid code to the standard 20 amino acid code with stops. The proposed patterns of code sectoring are now most apparent from patterns of aminoacyl-tRNA synthetase evolution. The Elongation Factor-Tu GTPase anticodon-codon latch that checks the accuracy of translation appears to have evolved at about the eight amino acid to ~16 amino acid stage. Before evolution of the EF-Tu latch, we posit that both the 1st and 3rd anticodon positions were wobble positions. The genetic code evolved via tRNA charging errors and via enzymatic modifications of amino acids joined to tRNAs, followed by tRNA and aminoacyl-tRNA synthetase differentiation. Fidelity mechanisms froze the code by inhibiting further innovation.

scholarly journals Phagosomal Neutralization by the Fungal Pathogen Candida albicans Induces Macrophage Pyroptosis

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
Slavena Vylkova ◽  
Michael C. Lorenz
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Amino Acid ◽  
Content Type ◽  
Caspase 1 ◽  
Ammonia Release ◽  
Hyphal Morphogenesis

ABSTRACT The interaction of Candida albicans with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that C. albicans co-opts amino acid catabolism to generate and excrete ammonia, which raises the extracellular pH, both in vitro and in vivo and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking STP2, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungus-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking AHR1 were defective in growth, alkalinization, and ammonia release on amino acid-rich media, similar to stp2Δ and ahr1Δ stp2Δ cells, and occupied more acidic phagosomes. Notably, ahr1Δ and stp2Δ strains did not induce pyroptosis, as measured by caspase-1-dependent interleukin-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that C. albicans-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of caspase-1-mediated macrophage lysis.

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