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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Article
  • Peer Reviewed
UC Irvine Previously Published Works (2014)
Epitope tagging of genes is a powerful technique facilitating assays for gene function, determination of subcellular distribution of proteins, affinity purification, study of protein interaction with other proteins, DNA or RNA, and any other antibody-based approach in the absence of protein-specific antibodies. Here, we describe a one-step PCR-based strategy for insertion of epitope tags at the chromosomal locus. This method takes advantage of efficient homologous recombination in yeast. PCR amplified tags are directed to desired chromosomal loci with the help of primer-encoded flanking homologous sequences enabling selective epitope tagging of genes of interest.
    Cover page: PCR-Mediated Epitope Tagging of Genes in YeastCreative Commons 'BY' version 4.0 license
    • Thesis
    • Peer Reviewed
    UC Irvine Electronic Theses and Dissertations (2014)

    Post-translational modification of proteins with ubiquitin plays a role in most biological processes. The key players of this system are the E3 ubiquitin ligases, which mediate substrate specific covalent attachment of ubiquitin. The SCF ubiquitin ligases are amongst best-understood E3 complexes. They are composed of Skp1, Cdc53/Cullin, Rbx1, and one of the multiple F-box proteins, which bind substrates and confer specificity to this complex. As these modular ligases share the same core components but differ in the identity of F-box proteins, the repertoire of SCF ligases present in a cell at any given time is thus governed by the abundance of different F-box proteins. Regulating F-box protein abundance is thus essential for normal cellular function and misregulation causes human diseases. In the studies presented here, we describe a novel mechanism governing maintenance of F-box protein homeostasis.

    In the absence of substrates, the core SCF ligase ubiquitylates its F-box protein and targets it for degradation. Using the yeast F-box protein Met30 as a model system, we demonstrate presence of an additional mechanism for F-box protein degradation apart from autoubiquitylation. This pathway targets F-box proteins that are dissociated from Skp1, and plays a crucial role in limiting substrate shielding effects caused by association of excess F-box proteins with their substrates thereby inhibiting substrate recognition by fully assembled ligases. Characterization of this pathway shows that it is governed by the ubiquitin proteasome system and is mediated by a ligase dependent on Cdc53 but independent of Skp1 and for the first time illustrates uncoupling of Cdc53 and Skp1 function in yeast.

    Furthermore, we show that Cic1 and the AAA ATPase Cdc48, along with its substrate recruiting factors Npl4/Ufd1 are involved in this mechanism of F-box protein degradation. Preliminary studies conducted to ascribe a role for these proteins in this process, suggest Cic1 as a unique factor that recruits ubiquitylated F-box proteins to Cdc48/Npl4/Ufd1 for targeting to proteasome for degradation.

    Together, our findings shed light on the complexity of the ubiquitin system. They uncover a novel degradation pathway, suggest existence of yet unknown types of ligases, and introduce the concept of protein specificity factors for Cdc48.

      Cover page: Identification of a Novel Mechanism for Regulation of F-box Proteins
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2015)
      Abundance of substrate receptor subunits of Cullin-RING ubiquitin ligases (CRLs) is tightly controlled to maintain the full repertoire of CRLs. Unbalanced levels can lead to sequestration of CRL core components by a few overabundant substrate receptors. Numerous diseases, including cancer, have been associated with misregulation of substrate receptor components, particularly for the largest class of CRLs, the SCF ligases. One relevant mechanism that controls abundance of their substrate receptors, the F-box proteins, is autocatalytic ubiquitylation by intact SCF complex followed by proteasome-mediated degradation. Here we describe an additional pathway for regulation of F-box proteins on the example of yeast Met30. This ubiquitylation and degradation pathway acts on Met30 that is dissociated from Skp1. Unexpectedly, this pathway required the cullin component Cdc53/Cul1 but was independent of the other central SCF component Skp1. We demonstrated that this non-canonical degradation pathway is critical for chromosome stability and effective defense against heavy metal stress. More importantly, our results assign important biological functions to a sub-complex of cullin-RING ligases that comprises Cdc53/Rbx1/Cdc34, but is independent of Skp1.
        Cover page: Skp1 Independent Function of Cdc53/Cul1 in F-box Protein Homeostasis
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2020)
        Organisms can adapt to a broad spectrum of sudden and dramatic changes in their environment. These abrupt changes are often perceived as stress and trigger responses that facilitate survival and eventual adaptation. The ubiquitin-proteasome system (UPS) is involved in most cellular processes. Unsurprisingly, components of the UPS also play crucial roles during various stress response programs. The budding yeast SCFMet30 complex is an essential cullin-RING ubiquitin ligase that connects metabolic and heavy metal stress to cell cycle regulation. Cadmium exposure results in the active dissociation of the F-box protein Met30 from the core ligase, leading to SCFMet30 inactivation. Consequently, SCFMet30 substrate ubiquitylation is blocked and triggers a downstream cascade to activate a specific transcriptional stress response program. Signal-induced dissociation is initiated by autoubiquitylation of Met30 and serves as a recruitment signal for the AAA-ATPase Cdc48/p97, which actively disassembles the complex. Here we show that the UBX cofactor Shp1/p47 is an additional key element for SCFMet30 disassembly during heavy metal stress. Although the cofactor can directly interact with the ATPase, Cdc48 and Shp1 are recruited independently to SCFMet30 during cadmium stress. An intact UBX domain is crucial for effective SCFMet30 disassembly, and a concentration threshold of Shp1 recruited to SCFMet30 needs to be exceeded to initiate Met30 dissociation. The latter is likely related to Shp1-mediated control of Cdc48 ATPase activity. This study identifies Shp1 as the crucial Cdc48 cofactor for signal-induced selective disassembly of a multisubunit protein complex to modulate activity.
          Cover page: Cdc48 cofactor Shp1 regulates signal-induced SCFMet30 disassembly
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2022)
          Telomerase activation counteracts senescence and telomere erosion caused by uncontrolled proliferation. Epidermal growth factor receptor (EGFR) amplification drives proliferation while telomerase reverse transcriptase promoter (TERTp) mutations underlie telomerase reactivation through recruitment of GA-binding protein (GABP). EGFR amplification and TERTp mutations typically co-occur in glioblastoma, the most common and aggressive primary brain tumor. To determine if these two frequent alterations driving proliferation and immortality are functionally connected, we combine analyses of copy number, mRNA, and protein data from tumor tissue with pharmacologic and genetic perturbations. We demonstrate that proliferation arrest decreases TERT expression in a GABP-dependent manner and elucidate a critical proliferation-to-immortality pathway from EGFR to TERT expression selectively from the mutant TERTp through activation of AMP-mediated kinase (AMPK) and GABP upregulation. EGFR-AMPK signaling promotes telomerase activity and maintains telomere length. These results define how the tumor cell immortality mechanism keeps pace with persistent oncogene signaling and cell cycling.
            Cover page: GABP couples oncogene signaling to telomere regulation in TERT promoter mutant cancer
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2020)

            Background

            Emerging data suggest that a subset of patients with diffuse isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG) who receive adjuvant temozolomide (TMZ) recur with hypermutation in association with malignant progression to higher-grade tumors. It is currently unclear why some TMZ-treated LGG patients recur with hypermutation while others do not. MGMT encodes O6-methylguanine-DNA methyltransferase, a DNA repair protein that removes cytotoxic and potentially mutagenic lesions induced by TMZ. Here, we hypothesize that epigenetic silencing of MGMT by promoter methylation facilitates TMZ-induced mutagenesis in LGG patients and contributes to development of hypermutation at recurrence.

            Methods

            We utilize a quantitative deep sequencing assay to characterize MGMT promoter methylation in 109 surgical tissue specimens from initial tumors and post-treatment recurrences of 37 TMZ-treated LGG patients. We utilize methylation arrays to validate our sequencing assay, RNA sequencing to assess the relationship between methylation and gene expression, and exome sequencing to determine hypermutation status.

            Results

            Methylation level at the MGMT promoter is significantly higher in initial tumors of patients that develop hypermutation at recurrence relative to initial tumors of patients that do not (45.7% vs 34.8%, P = 0.027). Methylation level in initial tumors can predict hypermutation at recurrence in univariate models and multivariate models that incorporate patient age and molecular subtype.

            Conclusions

            These findings reveal a mechanistic basis for observed differences in patient susceptibility to TMZ-driven hypermutation. Furthermore, they establish MGMT promoter methylation level as a potential biomarker to inform clinical management of LGG patients, including monitoring and treatment decisions, by predicting risk of hypermutation at recurrence.
              Cover page: MGMT promoter methylation level in newly diagnosed low-grade glioma is a predictor of hypermutation at recurrence
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