Hsp31, A Member of the DJ-1 Superfamily, is a multitaskingstress responder with chaperone activity

Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson’s disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including a-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson’s disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31’s chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purifed from yeast is more active compared to expression and purifcation from E. coli suggesting that posttranslational modifcations could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses

The Small Heat Shock Protein Hsp31 Cooperates with Hsp104 to Modulate Sup35 Prion Aggregation

The yeast homolog of DJ-1, Hsp31, is a multifunctional protein that is involved in several cellular pathways including detoxification of the toxic metabolite methylglyoxal and as a protein deglycase. Prior studies ascribed Hsp31 as a molecular chaperone that can inhibit a-Syn aggregation in vitro and alleviate its toxicity in vivo. It was also shown that Hsp31 inhibits Sup35 aggregate formation in yeast, however, it is unknown if Hsp31 can modulate [PSIC] phenotype and Sup35 prionogenesis. Other small heat shock proteins, Hsp26 and Hsp42 are known to be a part of a synergistic proteostasis network that inhibits Sup35 prion formation and promotes its disaggregation. Here, we establish that Hsp31 inhibits Sup35 [PSIC] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 transiently prevents prion induction but does not suppress induction upon prolonged expression of Sup35 indicating that Hsp31 can be overcome by larger aggregates. In addition, elevated levels of Hsp31 do not cure [PSIC] strains indicating that Hsp31 cannot intervene in a pre-existing prion oligomerization cycle. However, Hsp31 can modulate prion status in cooperation with Hsp104 because it inhibits Sup35 aggregate formation and potentiates [PSIC] prion curing upon overexpression of Hsp104. The absence of Hsp31 reduces [PSIC] prion curing by Hsp104 without influencing its ability to rescue cellular thermotolerance. Hsp31 did not synergize with Hsp42 to modulate the [PSIC] phenotype suggesting that both proteins act on similar stages of the prion cycle. We also showed that Hsp31 physically interacts with Hsp104 and together they prevent Sup35 prion toxicity to greater extent than if they were expressed individually. These results elucidate a mechanism for Hsp31 on prion modulation that suggest it acts at a distinct step early in the Sup35 aggregation process that is different from Hsp104. This is the first demonstration of the modulation of [PSIC] status by the chaperone action of Hsp31. The delineation of Hsp31’s role in the chaperone cycle has implications for understanding the role of the DJ-1 superfamily in controlling misfolded proteins in neurodegenerative disease and cancer

Inferring Gene RegulatoryNetworks from a Populationof Yeast Segregants

Constructing gene regulatory networks is crucial to unraveling the genetic architecture of complex traits and to understanding the mechanisms of diseases. On the basis of gene expression and single nucleotide polymorphism data in the yeast, Saccharomyces cerevisiae, we constructed gene regulatory networks using a two-stage penalized least squares method. A large system of structural equations via optimal prediction of a set of surrogate variables was established at the first stage, followed by consistent selection of regulatory effects at the second stage. Using this approach, we identified subnetworks that were enriched in gene ontology categories, revealing directional regulatory mechanisms controlling these biological pathways. Our mapping and analysis of expression-based quantitative trait loci uncovered a known alteration of gene expression within a biological pathway that results in regulatory effects on companion pathway genes in the phosphocholine network. In addition, we identify nodes in these gene ontology-enriched subnetworks that are coordinately controlled by transcription factors driven by trans-acting expression quantitative trait loci. Altogether, the integration of documented transcription factor regulatory associations with subnetworks defined by a system of structural equations using quantitative trait loci data is an effective means to delineate the transcriptional control of biological pathways

Sumoylation of The Budding Yeast Kinetochore Protein Ndc10 is Required for Ndc10 Spindle Localization and Regulation of Anaphase Spindle Elongation

Posttranslational modification by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) is emerging as an important regulator in many cellular processes, including genome integrity. In this study, we show that the kinetochore proteins Ndc10, Bir1, Ndc80, and Cep3, which mediate the attachment of chromosomes to spindle microtubules, are sumoylated substrates in budding yeast. Furthermore, we show that Ndc10, Bir1, and Cep3 but not Ndc80 are desumoylated upon exposure to nocodazole, highlighting the possibility of distinct roles for sumoylation in modulating kinetochore protein function and of a potential link between the sumoylation of kinetochore proteins and mitotic checkpoint function. We find that lysine to arginine mutations that eliminate the sumoylation of Ndc10 cause chromosome instability, mislocalization of Ndc10 from the mitotic spindle, abnormal anaphase spindles, and a loss of Bir1 sumoylation. These data suggest that sumoylation of Ndc10 and other kinetochore proteins play a critical role during the mitotic process

Antibiotic-induced Decreases inthe Levels of Microbial-derivedShort-chain Fatty Acids Correlatewith Increased GastrointestinalColonization of Candida Albicans

Candida albicans is the fourth most common cause of systemic nosocomial infections, posing a significant risk in immunocompromised individuals. As the majority of systemic C. albicans infections stem from endogenous gastrointestinal (GI) colonization, understanding the mechanisms associated with GI colonization is essential in the development of novel methods to prevent C. albicans-related mortality. In this study, we investigated the role of microbial-derived short-chain fatty acids (SCFAs) including acetate, butyrate, and propionate on growth, morphogenesis, and GI colonization of C. albicans. Our results indicate that cefoperazone-treated mice susceptible to C. albicans infection had significantly decreased levels of SCFAs in the cecal contents that correlate with a higher fungal load in the feces. Further, using in vivo concentration of SCFAs, we demonstrated that SCFAs inhibit the growth, germ tube, hyphae and biofilm development of C. albicans in vitro. Collectively, results from this study suggest that antibiotic-induced decreases in the levels of SCFAs in the cecum enhances the growth and GI colonization of C. albicans

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1CENP-T, the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn160–84, should be extended with flanking residues, Cnn125–91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1–S74, based on in vitro experiments demonstrating the Cnn1–Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase

Identification of a PhenylthiazoleSmall Molecule with DualAntifungal and Antibiofilm ActivityAgainst Candida albicans andCandida auris

Candida species are a leading source of healthcare infections globally. The limited number of antifungal drugs combined with the isolation of Candida species, namely C. albicans and C. auris, exhibiting resistance to current antifungals necessitates the development of new therapeutics. The present study tested 85 synthetic phenylthiazole small molecules for antifungal activity against drug-resistant C. albicans. Compound 1 emerged as the most potent molecule, inhibiting growth of C. albicans and C. auris strains at concentrations ranging from 0.25–2μg/mL. Additionally, compound 1 inhibited growth of other clinically-relevant yeast (Cryptococcus) and molds (Aspergillus) at a concentration as low as 0.50μg/mL. Compound 1 exhibited rapid fungicidal activity, reducing the burden of C. albicans and C. auris below the limit of detection within 30 minutes. Compound 1 exhibited potent antibiofilm activity, similar to amphotericin B, reducing the metabolic activity of adherent C. albicans and C. auris biofilms by more than 66% and 50%, respectively. Furthermore, compound 1 prolonged survival of Caenorhabditis elegans infected with strains of C. albicans and C. auris, relative to the untreated control. The present study highlights phenylthiazole small molecules, such as compound 1, warrant further investigation as novel antifungal agents for drug-resistant Candida infections

Inferring Gene Regulatory Networks from a Population of Yeast Segregants

Constructing gene regulatory networks is crucial to unraveling the genetic architecture of complex traits and to understanding the mechanisms of diseases. On the basis of gene expression and single nucleotide polymorphism data in the yeast, Saccharomyces cerevisiae, we constructed gene regulatory networks using a two-stage penalized least squares method. A large system of structural equations via optimal prediction of a set of surrogate variables was established at the first stage, followed by consistent selection of regulatory effects at the second stage. Using this approach, we identified subnetworks that were enriched in gene ontology categories, revealing directional regulatory mechanisms controlling these biological pathways. Our mapping and analysis of expression-based quantitative trait loci uncovered a known alteration of gene expression within a biological pathway that results in regulatory effects on companion pathway genes in the phosphocholine network. In addition, we identify nodes in these gene ontology-enriched subnetworks that are coordinately controlled by transcription factors driven by trans-acting expression quantitative trait loci. Altogether, the integration of documented transcription factor regulatory associations with subnetworks defined by a system of structural equations using quantitative trait loci data is an effective means to delineate the transcriptional control of biological pathways

Ospemifene Displays Broad-Spectrum Synergistic Interactions with Itraconazole Through Potent Interference with Fungal Efflux Activities

Azole antifungals are vital therapeutic options for treating invasive mycotic infections. However, the emergence of azole-resistant isolates combined with limited therapeutic options presents a growing challenge in medical mycology. To address this issue, we utilized microdilution checkerboard assays to evaluate nine stilbene compounds for their ability to interact synergistically with azole drugs, particularly against azole-resistant fungal isolates. Ospemifene displayed the most potent azole chemosensitizing activity, and its combination with itraconazole displayed broad-spectrum synergistic interactions against Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus (ΣFICI = 0.05–0.50). Additionally, in a Caenorhabditis elegans infection model, the ospemifene-itraconazole combination significantly reduced fungal CFU burdens in infected nematodes by ~75–96%. Nile Red efflux assays and RT-qPCR analysis suggest ospemifene interferes directly with fungal efflux systems, thus permitting entry of azole drugs into fungal cells. This study identifies ospemifene as a novel antifungal adjuvant that augments the antifungal activity of itraconazole against a broad range of fungal pathogens

Repurposing Approach IdentifiesPitavastatin as a Potent AzoleChemosensitizing Agent EffectiveAgainst Azole-Resistant CandidaSpecies

The limited number of antifungals and the rising frequency of azole-resistant Candida species are growing challenges to human medicine. Drug repurposing signifies an appealing approach to enhance the activity of current antifungal drugs. Here, we evaluated the ability of Pharmakon 1600 drug library to sensitize an azole-resistant Candida albicans to the effect of fluconazole. The primary screen revealed 44 non-antifungal hits were able to act synergistically with fluconazole against the test strain. Of note, 21 compounds, showed aptness for systemic administration and limited toxic effects, were considered as potential fluconazole adjuvants and thus were termed as “repositionable hits”. A follow-up analysis revealed pitavastatin displaying the most potent fluconazole chemosensitizing activity against the test strain (ΣFICI 0.05) and thus was further evaluated against 18 isolates of C. albicans (n = 9), C. glabrata (n = 4), and C. auris (n = 5). Pitavastatin displayed broad-spectrum synergistic interactions with both fluconazole and voriconazole against ~89% of the tested strains (ΣFICI 0.05–0.5). Additionally, the pitavastatin-fluconazole combination significantly reduced the biofilm-forming abilities of the tested Candida species by up to 73%, and successfully reduced the fungal burdens in a Caenorhabditis elegans infection model by up to 96%. This study presents pitavastatin as a potent azole chemosensitizing agent that warrant further investigation