Analysis of Kinase Signaling Pathways Regulating Filamentous Development in Saccharomyces cerevisiae and Candida albicans

Yeast pseudohyphal growth is a stress response characterized by elongated cell morphology, exaggerated polarized growth, and increased cell-cell adhesion. The signaling network that regulates the formation of pseudohyphal filaments has been the subject of intense research interest, as filament formation is required for virulence in numerous pathogenic fungi. Pseudohyphal growth is regulated through highly conserved kinase pathways, encompassing MAPK/ERK, PKA, and AMPK signaling modules; however, the full scope of these pathways has not been elucidated fully. To address this knowledge gap, quantitative phosphoproteomics was used in Saccharomyces cerevisiae to identify differentially phosphorylated proteins in kinase-deficient mutant strains surveyed under conditions inducing pseudohyphal growth. The use of stable isotope labeling of amino acids in cell culture (SILAC) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis of phosphopeptide-enriched samples, identified 439 phosphoproteins and 539 novel phosphorylated residues dependent on a pseudohyphal growth kinase. This data set yielded two interesting results. First, the identified phosphoprotein set was significantly enriched for ribonucleoprotein (RNP) granule components, including P-bodies and stress granules. Through fluorescence microscopy of GFP-chimeras, co-localization of the MAPK cascade proteins Kss1p, Ste20p, Fus3p, and also PKA (Tpk2p) with the RNP component Igo1p was observed. Furthermore, Kss1p kinase activity was required for wild-type levels of mRNA localization in P-bodies. Second, the phosphoproteomic data set indicated that a statistically significant set of kinases mediating inositol polyphosphate (IP) signaling undergoes pseudohyphal growth kinase-dependent phosphorylation. Deletion of kinases in the IP synthesis pathway resulted in aberrant pseudohyphal growth, and through metabolic labeling of IP species, striking changes in IP levels under pseudohyphal growth conditions were observed. In particular, a correlation between increased filamentous growth and the presence of high levels of the inositol pyrophosphate produced by Kcs1p, 5PP-IP5 were found. Additionally, interesting changes in IP levels upon the deletion of key kinases involved in the pseudohyphal growth transition were observed. With the relevance of yeast pseudohyphal growth as a model of filamentous development in the pathogen Candida albicans, these studies investigated the role of the C. albicans cell wall integrity kinase Cbk1p in morphogenesis during hyphal development. In collaborative studies with Damian Krysan’s group at the University of Rochester, this work indicated a mechanism where CaCbk1p regulates cross-talk between the RAM and PKA pathways. Taken together, the doctoral research presented here provides insight into the mechanisms through which conserved kinase signaling networks regulate filamentous growth, with particular relevance in understanding the mechanisms controlling RNP dynamics and regulated IP biogenesis.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140976/1/knorm_1.pd

Identifying Signaling Pathways Mediating Filamentous Growth in Saccharomyces Cerevisiae.

Multiple fungal species exhibit complex morphological changes in response to environmental conditions. Notably, the ability to transition between a cellular yeast-like form and a filamentous form is linked to virulence in several plant and human pathogens. My research is focused on identifying the molecular mechanisms driving this dimorphism in budding yeast. In S. cerevisiae, nutrient stress can induce morphological changes where unicellular yeast transition to a filamentous state, marked by pseudohyphal filaments of elongated and connected cells. This pseudohyphal differentiation is regulated by core signaling pathways responsive to diverse environmental stimuli, but the complete repertoire of molecular components coordinating these signals is unclear. To identify novel regulators of yeast stress-responsive pseudohyphal growth, I leveraged an innovative bioinformatics tool to discover biologically meaningful relationships from high-throughput data. Specifically, I analyzed publicly available and novel DNA microarray data sets using the Topology Enrichment Analysis framework (TEAK) and identified two previously unreported genes necessary for the yeast nitrogen stress response (DPL1 and LAG1) as well as a key regulator of lipid metabolism (SLC1) that is required for pseudohyphal differentiation. Separately, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen stress. To identify the Sks1p signaling network, we applied mass spectrometry-based phosphoproteomics and identified over 900 phosphosites that exhibited Sks1p kinase-dependent changes. From this analysis, we report a set of novel phosphosites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2. Additionally, the pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/102424/1/colejohn_1.pd

Identification and characterization of novel putative virulence factors in Candida albicans

The C. albicans community is currently laying the foundation of understanding how this human pathogen causes infection. C. albicans infections represent a major medical and economic burden for today’s society with an estimated 400,000 blood stream infections worldwide and direct costs exceeding 1$ billion dollar a year in the U.S. alone. Although finding the biological causes of this disease seemed to be beyond our reach in the past, various aspects of the infection have been recently unveiled including its pathology, immunology, histology, and epidemiology. Here we explored the genetic components of this disease by studying the complex host-pathogen dynamics through a series of in vivo, ex vivo and in vitro experiments. By using a pathogen unbiased reverse genetic approach and a host gene candidate strategy we uncovered some of the genes and pathways that are important for pathogenicity and immunity. In particular we explored the complex host-pathogen dynamics using a C. albicans - C. elegans model system and identified four novel putative virulence factors. We focused on Zcf15, a C. albicans transcription factor that has been poorly characterized in the literature and that plays an important role in the pathogen’s ability to resist host generated reactive oxygen species (ROS). By leveraging the power of RNASeq and ChIP-Seq we identified Zcf15 transcriptional targets and DNA binding sites. These studies suggest that Zcf15 plays a critical role in carbon metabolism and that it exerts its ability to protect the pathogen from ROS by controlling the expression of thiol- peroxidases and other detoxifying enzymes. We also showed here that in C. elegans, the host’s ability to counteract the infection relies on the MAPK pathway, evidence that mirrors what has been found by others in mammals and that emphasizes the usefulness of studying C. albicans infections in smaller genetically traceable organisms like C. elegans. The nematode model is also shown here to be a powerful tool not only to study the genetic bases that drive infection and immunity but also to identify new compounds that can be used for therapeutic intervention. This model was instrumental in identifying filastatin, a small molecule that was subsequently found by our collaborators to be capable of reducing virulence in mammals. The antifungal properties of filastatin are currently undertaking further preclinical testing. Overall this thesis shed light on the complex mechanisms of C. albicans pathogenicity and host immunity and identified novel virulence determinants that can be used by the larger community for further biological studies or even drug development

A constraint optimization framework for discovery of cellular signaling and regulatory networks

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Cellular signaling and regulatory networks underlie fundamental biological processes such as growth, differentiation, and response to the environment. Although there are now various high-throughput methods for studying these processes, knowledge of them remains fragmentary. Typically, the majority of hits identified by transcriptional, proteomic, and genetic assays lie outside of the expected pathways. In addition, not all components in the regulatory networks can be exposed in one experiment because of systematic biases in the assays. These unexpected and hidden components of the cellular response are often the most interesting, because they can provide new insights into biological processes and potentially reveal new therapeutic approaches. However, they are also the most difficult to interpret. We present a technique, based on the Steiner tree problem, that uses a probabilistic protein-protein interaction network and high confidence measurement and prediction of protein-DNA interactions, to determine how these hits are organized into functionally coherent pathways, revealing many components of the cellular response that are not readily apparent in the original data. We report the results of applying this method to (1) phosphoproteomic and transcriptional data from the pheromone response in yeast, and (2) phosphoproteomic, DNaseI hypersensitivity sequencing and mRNA profiling data from the U87MG glioblastoma cell lines over-expressing the variant III mutant of the epidermal growth factor receptor (EGFRvIII). In both cases the method identifies changes in diverse cellular processes that extend far beyond the expected pathways. Analysis of the EGFRVIII network connectivity property and transcriptional regulators that link observed changes in protein phosphorylation and differential expression suggest a few intriguing hypotheses that may lead to improved therapeutic strategy for glioblastoma.by Shao-shan Carol Huang.Ph.D

Ubiquitination in Health and Diseases

Ubiquitination is a biological process mediated by ubiquitin itself, the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, E3 ubiquitin ligase, and deubiquitinating enzyme, respectively. Currently, these multiple biological steps are revealed to participate in various life phenomena, such as cell proliferation, regulation of cell surface proteins expression, and mitochondrial function, which are profoundly related to human health and diseases. Although clinical applications targeting ubiquitination are still limited compared to those directed toward kinase systems such as tyrosine kinases, multiple enzymatic consequences should be future therapeutic implications. This Special Issue of IJMS entitled “Ubiquitination in Health and Disease” successfully published15 distinguished manuscripts, with a total of 66 international authors and. This book provides the latest and most useful information for researchers and scientists in this field

Functionally Relevant Macromolecular Interactions of Disordered Proteins

Disordered proteins are relatively recent newcomers in protein science. They were first described in detail by Wright and Dyson, in their J. Mol. Biol. paper in 1999. First, it was generally thought for more than a decade that disordered proteins or disordered parts of proteins have different amino acid compositions than folded proteins, and various prediction methods were developed based on this principle. These methods were suitable for distinguishing between the disordered (unstructured) and structured proteins known at that time. In addition, they could predict the site where a folded protein binds to the disordered part of a protein, shaping the latter into a well-defined 3D structure. Recently, however, evidence has emerged for a new type of disordered protein family whose members can undergo coupled folding and binding without the involvement of any folded proteins. Instead, they interact with each other, stabilizing their structure via “mutual synergistic folding” and, surprisingly, they exhibit the same residue composition as the folded protein. Increasingly more examples have been found where disordered proteins interact with non-protein macromolecules, adding to the already large variety of protein–protein interactions. There is also a very new phenomenon when proteins are involved in phase separation, which can represent a weak but functionally important macromolecular interaction. These phenomena are presented and discussed in the chapters of this book

Therapeutic Potential of Plant Secondary Metabolites in the Treatment of Diseases and Drug Development

The importance of natural products, and especially plant secondary metabolites, for the treatment of diseases and drug development has already been obvious in medicine for several thousand years. Thus, this Special Issue of MDPI Biomedicines collects eight top articles from the field as regular full papers in addition to five reviews. All of the published papers are a vibrant source of information on the therapeutic potential of plant secondary metabolites in the treatment of diseases and drug development

Co-cultivation of non-conventional yeast with Saccharomyces cerevisiae to increase the aroma complexity of fermented beverages

Yeast are used as workhorses to convert hopped wort into beer. Conventionally, such yeasts belong to the genus Saccharomyces and most research on fermentation of wort for the production of beer has focussed on the species Saccharomyces cerevisiae and Saccharomyces pastorianus. Recently, there is an increasing interest in unravelling features of non-conventional yeast species for beer innovation. In this thesis, features of yeast isolates belonging to the species: Cyberlindnera fabianii, Pichia kudriavzevii and S. cerevisiae (all isolated from fermented masau (Ziziphus mauritiana) fruits in Zimbabwe), were studied with focus on aroma production. Additionally, a novel approach was used to apply these yeasts in co-cultivation with Brewers’ yeast (S. cerevisiae) for beer innovation. The characteristics and quality of the beer are mainly determined by aroma compounds in the final product such as esters, alcohols, aldehydes and acids. Yeast use various metabolic pathways such as glycolysis, the fermentative pathway, the tricarboxylic acid (TCA) cycle and the Ehrlich pathway to produce aroma compounds or the precursors for the synthesis thereof (Chapter 1). Among the aroma compounds, esters are of major importance, especially since they are perceived by the human olfactory system at very low concentrations. In general, esters are desirable compounds in beers due to their fruity flavour. Examples are isoamyl acetate (banana), isobutyl acetate (fruity, sweet), phenylethyl acetate (rose, apple, honey), ethyl acetate (sweet pear), ethyl hexanoate (apple, aniseed) and ethyl octanoate (sour apple). Together with an extensive range of other volatile organic compounds (VOCs) these compounds were previously profiled using headspace solid-phase-micro-extraction gas-chromatography mass-spectrometry (GCMS). Interestingly, comparative profiling of aromas showed that C. fabianii produces significantly higher amounts of isoamyl acetate and ethyl acetate compared to S. cerevisiae. It has been suggested in literature that products of the Ehrlich pathway, so called “fusel alcohols”, can function as signalling molecules for invasive growth upon nitrogen limitation. This suggested that nutrient limitation could affect growth performance and production of aroma compounds. Therefore, in Chapter 2, the metabolic and morphological response of S. cerevisiae, C. fabianii and P. kudriavzevii was analysed upon nitrogen and/or glucose limitation on semi-solid (agar) media. All three yeasts showed a change in growth mode upon nitrogen and/or glucose limitation. Scanning electron microscopy was used to unravel the cell organisation of C. fabianii and P. kudriavzevii grown under nitrogen limitation. This revealed the power of cell-cell adhesion for penetrative growth and the formation of meta-filaments or pseudo-hyphae to extend the cell surface area. Such changes in growth mode may be of relevance for solid-state fermentation processes, such as fruit fermentations, by enhanced substrate penetration of yeast. Notably, a significant increase in the production of esters (ethyl acetate, ethyl propanoate, isobutyl acetate and isopentyl acetate) by C. fabianii and P. kudriavzevii was found under conditions of nitrogen limitation. Understanding the relationship between nitrogen limitation and ester formation gives more insight into the ability to steer ester formation by nutrient availability in wort fermentations. The amount and type of esters are important determinants of the final flavour characteristics of the beer. Therefore, the diversity in ester production between the three yeast species was investigated by studying enzymatic reactions involved in the production (synthesis) of esters and their degradation (hydrolysis) and to link this to volatile organic compound profiles (Chapter 3). The amount and type of esters depends on substrate availability and a combination of enzyme activities supporting the synthesis and hydrolysis of the different esters formed. Esters are generally formed by a condensation reaction of an alcohol with ac(et)yl CoA by a so-called alcohol acetyltranferase (AATse). The products formed can generally be subdivided into two groups; acetate esters and medium chain fatty acid (MCFA) esters. Alcohols are formed by reduction of aldehydes by alcohol dehydrogenases (ADH) using either the fermentative or Ehrlich pathway. Comparative analysis of the specific ADHs and acetate ester hydrolysing activity and subsequently linking these data with the distinct aroma profiles of the three yeasts revealed that the acetate ester hydrolysing activity is a key step in determining the final pool size of acetate esters found in the fermentation broths (Chapter 3). Under the experimental conditions, C. fabianii showed the lowest acetate ester hydrolysing activity correlating with higher extracellular levels of acetate esters indicating the suitability of this yeast for use in co-cultures with brewers’ yeast with the objective to enable the enrichment of acetate esters in the fermentation process (Chapters 4 and 5). Nowadays, there is large interest of consumers in specialty beers such as beers low in alcohol (health awareness) and/or richer is fruity flavours (specialty beers), and this has significantly stimulated the quest for new methods, practices and yeast strains to produce such beers. In chapter 4 an innovative approach is described using co-cultivations of brewers’ yeast and C. fabianii to steer wort fermentation performance. Various ratios of brewers’ yeast over C. fabianii were inoculated in wort. A dose response relationship was observed, where a higher initial dose of C. fabianii leads to lower alcohol production and a more complex aroma bouquet. Interestingly, specific esters, i.e. ethyl acetate (sweet pear), ethyl octanoate (sour apple), ethyl decanoate (waxy, sweet apple), ethyl 9-decenoate (fruity, fatty) and ethyl dodecanoate (fruity, waxy), were found in higher levels in co-cultivation compared to both mono-cultivations indicating metabolic interactions. The reduced ethanol production in the co-culture could be explained by inhibition of Brewers’ yeast performance by C. fabianii in the co-cultivations. Further investigations revealed that this growth inhibition is caused by competition for oxygen between brewers’ yeast and C. fabianii. Depletion of oxygen caused inhibition of growth of brewers’ yeast since it needs oxygen to synthesize ergosterol that is required for cell membrane synthesis under anaerobic conditions (Chapter 4). The interaction between brewers’ yeast and C. fabianii in co-cultivation can be described using dynamic modelling (Chapter 5). A dynamic model was developed based on brewers’ yeast and C. fabianii in mono-cultivation and fitted to experimental data. The two models were combined and the same parameter settings were used to predict the fermentation outcome of brewers’ yeast and C. fabianii in co-cultivation. The model was experimentally validated using inoculation ratios of 1:10 and 1:100 brewers’ yeast over C. fabianii. Additionally, the use of dynamic modelling supported the hypothesis that competition for oxygen between brewers’ yeast and C. fabianii results in inhibition of brewers’ yeast fermentation performance. Interestingly, prediction of aroma formation in co-cultivation, especially that of specific esters, appeared to be more challenging due to metabolic interactions resulting in MCFA-esters contributing to fruity aromas, and this aspect requires further study. The results and findings obtained in the experimental chapters (Chapter 2-5) are further discussed in Chapter 6. Unravelling features of non-conventional yeast generates novel opportunities for beer innovation. Application of C. fabianii in co-cultivation with brewers’ yeast in wort fermentation offers a novel approach in product innovation resulting in low alcohol beers with enriched aroma bouquet . Finally, the developed dynamic model may be used to predict fermentation outcomes of brewers’ yeast with other non-conventional yeast species.</p

Molecular Pathways in Cancers

This book includes some recent works providing the readers with novel relevant findings about the main signaling pathways that govern the molecular pathogenesis of some of the highest prevalent human tumors, which are the basis for developing alternative therapeutic strategies to improve patient outcomes

Program and Proceedings: The Nebraska Academy of Sciences 1880-2023. 142th Anniversary Year. One Hundred-Thirty-Third Annual Meeting April 21, 2023. Hybrid Meeting: Nebraska Wesleyan University & Online, Lincoln, Nebraska

AERONAUTICS & SPACE SCIENCE Chairperson(s): Dr. Scott Tarry & Michaela Lucas HUMANS PAST AND PRESENT Chairperson(s): Phil R. Geib & Allegra Ward APPLIED SCIENCE & TECHNOLOGY SECTION Chairperson(s): Mary Ettel BIOLOGY Chairpersons: Lauren Gillespie, Steve Heinisch, and Paul Davis BIOMEDICAL SCIENCES Chairperson(s): Annemarie Shibata, Kimberly Carlson, Joseph Dolence, Alexis Hobbs, James Fletcher, Paul Denton CHEM Section Chairperson(s): Nathanael Fackler EARTH SCIENCES Chairpersons: Irina Filina, Jon Schueth, Ross Dixon, Michael Leite ENVIRONMENTAL SCIENCE Chairperson: Mark Hammer PHYSICS Chairperson(s): Dr. Adam Davis SCIENCE EDUCATION Chairperson: Christine Gustafson 2023 Maiben Lecturer: Jason Bartz 2023 FRIEND OF SCIENCE AWARD TO: Ray Ward and Jim Lewi