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Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2014)

Guanine nucleotide binding proteins (G proteins) and their cell surface G protein coupled receptors (GPCR) regulate many cellular processes in filamentous fungi, including growth, sexual, and asexual development. Neurospora crassa (N. crassa) has three G alpha subunits (GNA-1, GNA-2 and GNA-3), one G beta subunit (GNB-1), and one G gamma subunit (GNG-1). In this study, the three N. crassa G alpha subunits are analyzed for genetic epistasis with G beta subunit. Using double mutants lacking one G alpha gene and gnb-1 and introduced constitutively active, GTPase-deficient alleles for each G alpha gene into the Δgnb-1 background genetic epistasis relationships were determined between G beta and G alpha subunit genes.

The length of the G protein signal is controlled by the duration of the GTP-bound alpha subunit; the intensity of the G protein signal is controlled by the level of activated G alpha subunits, which directly correlated with amount of protein and this regulation is not well known in filamentous fungi. Part of this dissertation focuses on investigation of the regulation of the G alpha subunits protein level, interaction with cytosolic proteins, translation, and degradation of G alpha subunit. Using endogenously tagged G alpha subunit (GNA-1), we identified cytosolic proteins associated with GNA-1. Putative GNA-1 interactors, which could affect stability of G alpha subunit, were checked for the level of the GNA-1 protein, including four 19S proteasome regulatory subunit deletion mutants. Our study shows that GNA-1 degraded by proteasome pathway. Level of G alpha subunit proteins in a whole cell extracts isolated from submerged liquid cultures are decreased in the Δgnb-1 and Δric8 strains relative to the wild type, with GNA-1 showing the greatest effect. Treatments with degradational inhibitors shows that protein turnover is not a mechanism responsible the low level of the G alpha subunit proteins in the Δgnb-1 and Δric8 backgrounds.

To determine if the translation is the regulatory mechanism of the G alpha proteins in the Δgnb-1 and Δric8 backgrounds, I stablished ribosomal profiling protocol for N. crassa. During adaptation of the protocol I found that cycloheximide pretreatment of living cells before harvesting and TRIzol extraction protocol are not suitable for N. crassa polysome-profiling protocol. I also found that GNB-1 and RIC8 protein associated with ribosomes or other elements of translational machinery.

    Cover page: Regulation of the G Protein Alpha Subunits in Mutants Lacking the G Beta Subunit or a Cytosolic Guanine Nucleotide Exchange Factor in Neurospora crassa.
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2019)
    G protein-coupled receptors (GPCRs) are targets for ∼35% of approved drugs but only ∼15% of the ∼800 human GPCRs are currently such targets. GPCRomics, the use of unbiased, hypothesis-generating methods [e.g., RNA-sequencing (RNA-seq)], with tissues and cell types to identify and quantify GPCR expression, has led to the discovery of previously unrecognized GPCRs that contribute to functional responses and pathophysiology and that may be therapeutic targets. The combination of GPCR expression data with validation studies (e.g., signaling and functional activities) provides opportunities for the discovery of disease-relevant GPCR targets and therapeutics. Here, we review insights from GPCRomic approaches, gaps in knowledge, and future directions by which GPCRomics can advance GPCR biology and the discovery of new GPCR-targeted drugs.
      Cover page: GPCRomics: An Approach to Discover GPCR Drug Targets
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2015)
      G protein-coupled receptors (GPCRs), the largest family of signaling receptors in the human genome, are also the largest class of targets of approved drugs. Are the optimal GPCRs (in terms of efficacy and safety) currently targeted therapeutically? Especially given the large number (∼ 120) of orphan GPCRs (which lack known physiologic agonists), it is likely that previously unrecognized GPCRs, especially orphan receptors, regulate cell function and can be therapeutic targets. Knowledge is limited regarding the diversity and identity of GPCRs that are activated by endogenous ligands and that native cells express. Here, we review approaches to define GPCR expression in tissues and cells and results from studies using these approaches. We identify problems with the available data and suggest future ways to identify and validate the physiologic and therapeutic roles of previously unrecognized GPCRs. We propose that a particularly useful approach to identify functionally important GPCRs with therapeutic potential will be to focus on receptors that show selective increases in expression in diseased cells from patients and experimental animals.
        Cover page: G Protein–Coupled Receptor (GPCR) Expression in Native Cells: “Novel” endoGPCRs as Physiologic Regulators and Therapeutic Targets
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2015)
        G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization.
          Cover page: Global Analysis of Predicted G Protein−Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2015)
          G protein-coupled receptors (GPCRs) regulate facets of growth, development, and environmental sensing in eukaryotes, including filamentous fungi. The largest predicted GPCR class in these organisms is the Pth11-related, with members similar to a protein required for disease in the plant pathogen Magnaporthe oryzae. However, the Pth11-related class has not been functionally studied in any filamentous fungal species. Here, we analyze phenotypes in available mutants for 36 GPCR genes, including 20 Pth11-related, in the model filamentous fungus Neurospora crassa. We also investigate patterns of gene expression for all 43 predicted GPCR genes in available datasets. A total of 17 mutants (47%) possessed at least one growth or developmental phenotype. We identified 18 mutants (56%) with chemical sensitivity or nutritional phenotypes (11 uniquely), bringing the total number of mutants with at least one defect to 28 (78%), including 15 mutants (75%) in the Pth11-related class. Gene expression trends for GPCR genes correlated with the phenotypes observed for many mutants and also suggested overlapping functions for several groups of co-transcribed genes. Several members of the Pth11-related class have phenotypes and/or are differentially expressed on cellulose, suggesting a possible role for this gene family in plant cell wall sensing or utilization.
            Cover page: Global Analysis of Predicted G Protein-Coupled Receptor Genes in the Filamentous Fungus, Neurospora crassa.
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