| 1. |
- Alvarez, Francisco J., et al.
(author)
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Diverse Nitrogen Sources in Seminal Fluid Act in Synergy To Induce Filamentous Growth of Candida albicans
- 2015
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 81:8, s. 2770-2780
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Journal article (peer-reviewed)abstract
- The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.
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| 2. |
- Boban, Mirta, et al.
(author)
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Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation
- 2015
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:4, s. 2489-2495
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Journal article (peer-reviewed)abstract
- Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to epsilon-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.
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| 3. |
- Llopis-Torregrosa, Vicent, et al.
(author)
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Trk1-mediated potassium uptake contributes to cell-surface properties and virulence of Candida glabrata
- 2019
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Journal article (peer-reviewed)abstract
- The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1 Delta cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1 Delta mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.
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| 4. |
- Martins, António, et al.
(author)
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Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor-controlled Ssy5 signaling protease
- 2019
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 30:21, s. 2709-2720
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Journal article (peer-reviewed)abstract
- The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5) sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy terminus of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Delta tether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution, and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non-and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.
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| 5. |
- Martins, António, et al.
(author)
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Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity
- 2018
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In: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 293:22, s. 8362-8378
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Journal article (peer-reviewed)abstract
- Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.
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| 6. |
- Martins, António, 1976-
(author)
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The cell biology and catalytic properties of the nutrient-induced signaling endoprotease Ssy5
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Cells continuously sense and respond to changes in the presence, quality and quantity of external and internal nutrients. Specific signaling proteases have been identified based on their roles in processing or destruction of distinct sets of downstream effector proteins in response to environmental cues. The Saccharomyces cerevisiae Ssy5 signaling endoprotease has a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival. Ssy5 is a core component of the Ssy1–Ptr3-Ssy5 (SPS) sensor, which enables yeast cells to respond to extracellular amino acids and induce their uptake. Ssy5 cleaves transcription factors Stp1 and Stp2, permitting their translocation to the nucleus where they enhance the expression of amino acid permease genes. This thesis focuses on Ssy5, its biogenesis and catalytic properties (paper I), the spatial determinants underlying Ssy5 function in SPS-sensor context (paper II) and substrate cleavage (paper III).Ssy5 is comprised of pro- and catalytic-(Cat)-domains. The Cat-domain possesses characteristic hallmarks of a serine protease; however, serine protease-specific inhibitors have limited effect, confounding its classification. In paper I we unambiguously show that Ssy5 is a serine protease, define the precise sites of cleavage in Stp1 and Stp2, and describe the sequence specific requirements of their cleavage. The uniquely large prodomain (381 amino acids) has two essential functions. Initially, it is required in cis for the maturation of the Cat-domain, helping to overcome a folding barrier that is reflected in the high stability of the Cat-domain. Subsequent to attaining enzymatic competence, Ssy5 undergoes an autolytic cleavage event. The domains remain associated and the prodomain functions to fetter the proteolytic activity of the Cat-domain.The plasma membrane (PM) localization of Ssy1 has recently been questioned in a report that postulated that Ssy1 is a component of the endoplasmic reticulum (ER) and contributes to the formation of ER-PM junctions. In paper II, using mutational and subcellular fractionation experiments we critically examined this notion that is inconsistent with the current understanding of Ssy5 activation, i.e., the unfettering of the Cat-domain occurs in strict association with Ssy1 at the PM. The data show that Ssy1 is indeed a PM protein, and importantly, Ssy5-activation occurs independent of ER-PM junctions. A di-acidic ER exit motif was identified that is critical for proper PM localization and function of Ssy1. In paper III, we report that the Cat-domain is post-translationally modified in a manner dependent on Ptr3 and the PM casein kinase I (Yck1/2), consistent with Ssy5 activation occurring at the PM. Strikingly, the activated Cat-domain is capable of properly cleaving Stp1 fused to an ER membrane protein. The amino acid-induced cleavage of this artificial membrane-bound substrate occurs in a Δtether strain (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ) lacking ER-PM junctions. These findings indicate that the activated Cat-domain can bind and functionally interact with substrates distant from the PM. Finally, we show that the Cat-domain is degraded faster in amino acid-induced cells. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the resetting of the SPS-sensing system correlates with Cat-domain degradation.
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| 7. |
- Pantazopoulou, Marina, et al.
(author)
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Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation
- 2016
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 129:20, s. 3770-3780
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Journal article (peer-reviewed)abstract
- The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t(1/2)<= 30 min). Its turnover depends on ubiquitin-mediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.
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| 8. |
- Ring, Andreas, 1983-
(author)
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In vivo analysis of amino acid permease folding in yeast
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Plasma membrane (PM) proteins are critical for cells to respond to environmental cues, such as the availability of nutrients. The yeast Saccharomyces cerevisiae is able to sense extracellular amino acids using the SPS sensing system. Activation of the multimeric PM-localized SPS(Ssy1-Ptr3-Ssy5)-sensor complex occurs upon binding of external amino acids to Ssy1, inducing a conformational change. In a Ptr3-mediated event, the catalytic activity of the Ssy5 endoprotease is unfettered, leading to the proteolytic processing of two latent transcription factors, Stp1 and Stp2. Ssy1, the primary sensor component, is a non-transporting member of the amino acid permease (AAP) family of transport proteins, a family of eighteen complex integral membrane proteins comprised of 12 transmembrane segments (TMS). The AAPs exhibit a common requirement for the endoplasmic reticulum (ER)-localized membrane chaperone Shr3 to fold and to be transported to the PM. The absence of Shr3 leads to the accumulation of misfolded AAP species that are targeted for ER-associated degradation. Thus, proper Shr3 function is required as the most upstream and most downstream component of the SPS sensing system. In paper I, we investigate the chaperone function of Shr3. We report a surprisingly low level of sequence specificity underlies Shr3-AAP interactions. We used a split-ubiquitin approach to probe Shr3-AAP interactions in vivo. The Shr3-AAP interactions initiate early after the first two-to-four TMS of AAPs insert into the ER membrane, successively strengthening and then diminishing after all 12 TMS partition into the membrane. In paper II, we clarified the localization and trafficking determinants of Ssy1. A study by Kralt et al. 2015 reported that Ssy1 primarily localizes to the ER and is sorted to ER-PM tethers. These reported findings are clearly incompatible with the accepted model of amino acid sensing by the SPS-sensor. We critically re-examined the localization of Ssy1 and found that it indeed localizes to the PM, and importantly does so independent of ER-PM tethers. We also identified a novel ER exit motif in the carboxy-terminal tail of Ssy1 required for proper PM localization and SPS-sensor function. In paper III, we report that Ssy5 is able to cleave substrates in unusual contexts, i.e., an engineered substrate carrying rearranged recognition and cleavage determinants placed ectopically at the carboxy terminus of Stp1, and an ER-anchored substrate with Stp1 fused to the carboxy terminus of Shr3. Strikingly, Ssy5 catalyzed cleavage of Shr3-Stp1 in cells lacking ER-PM tethers, indicating that once activated, Ssy5 can find and cleave substrates distant from the PM. Consequently, cells must be able to rein in the activity of the Ssy5 protease to prevent spurious and improper proteolysis. Consistent with this notion, we report that the catalytic domain of Ssy5 is ubiquitylated in a Ptr3 and Yck1/2 dependent manner, and under amino acid-inducing conditions is subject to degradation. We propose a model that degradation of the Ssy5 catalytic domain is essential for resetting the SPS sensing system and a requisite for cells to regain the ability to correctly sense extracellular amino acids.
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| 9. |
- Ring, Andreas, et al.
(author)
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Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of plasma membrane‐endoplasmic reticulum junctions
- 2019
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In: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 20:10, s. 775-784
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Journal article (peer-reviewed)abstract
- Evidence from multiple laboratories have implicated Ssy1, a non‐transporting amino acid permease, as the receptor component of the yeast plasma membrane (PM)‐localized SPS (Ssy1‐Ptr3‐Ssy5)‐sensor. Upon binding external amino acids, Ssy1 is thought to initiate signaling events leading to the induction of amino acid permease gene expression. In striking contrast, Kralt et al. 2015 (Traffic 16:135‐147) have questioned the role of Ssy1 in amino acid sensing and reported that Ssy1 is a component of the endoplasmic reticulum (ER), where it reportedly participates in the formation of ER‐PM junctions. Here, we have re‐examined the intracellular location of Ssy1 and tested the role of ER‐PM junctions in SPS sensor signaling. We show that the C‐terminal of Ssy1 carries a functional ER‐exit motif required for proper localization of Ssy1 to the PM. Furthermore, ER‐PM junctions are dispensable for PM‐localization and function of Ssy1; Ssy1 localizes to the PM in a Δtether strain lacking ER‐PM junctions (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ), and this strain retains the ability to initiate signals induced by extracellular amino acids. The data demonstrate that Ssy1 functions as the primary amino acid receptor and that it carries out this function at the PM.
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| 10. |
- Silao, Fitz Gerald S., et al.
(author)
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Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans
- 2019
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In: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 15:2
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Journal article (peer-reviewed)abstract
- Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence. Author summary Candida albicans is an opportunistic fungal pathogen that exists as a benign member of the human microbiome. Immunosuppression, or microbial dysbiosis, can predispose an individual to infection, enabling this fungus to evade innate immune cells and initiate a spectrum of pathologies, including superficial mucocutaneous or even life-threatening invasive infections. Infectious growth is attributed to an array of virulence characteristics, a major one being the ability to switch morphologies from round yeast-like to elongated hyphal cells. Here we report that mitochondrial proline catabolism is required to induce hyphal growth of C. albicans cells in phagosomes of engulfing macrophages, which is key to evade killing by macrophages. The finding that proline catabolism, also required for the utilization of arginine and ornithine, is required to sustain the energy demands of hyphal growth underscores the central role of mitochondria in fungal virulence. In contrast to existing dogma, we show that in C. albicans, mitochondrial function is subject to glucose repression, amino acid-induced signals are strictly dependent on Ras1 and the SPS-sensor is the primary sensor of extracellular amino acids. The results provide a clear example of how C. albicans cells sense and respond to host nutrients to ensure proper nutrient uptake and survival.
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