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Open Access Publications from the University of California

Scholarly Works (84 results)

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The most prevalent cause of cystic fibrosis (CF) is the deletion of a phenylalanine residue at position 508 in CFTR (ΔF508-CFTR) protein. The mutated protein fails to fold properly, is retained in the endoplasmic reticulum via the action of molecular chaperones, and is tagged for degradation. In this study, the differences in protein expression levels in CF cell models were assessed using a systems biology approach aided by the sensitivity of MudPIT proteomics. Analysis of the differential proteome modulation without a priori hypotheses has the potential to identify markers that have not yet been documented. These may also serve as the basis for developing new diagnostic and treatment modalities for CF. Several novel differentially expressed proteins observed in our study are likely to play important roles in the pathogenesis of CF and may serve as a useful resource for the CF scientific community.
    Cover page: Quantitative Proteomic Profiling Reveals Differentially Regulated Proteins in Cystic Fibrosis Cells
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    • Peer Reviewed
    UC Irvine Previously Published Works (1994)
    Tuberous sclerosis (TSC) is an autosomal dominant condition with characteristic skin lesions, mental handicap, seizures and the development of hamartomas in the brain, heart, kidneys and other organs. Linkage studies have shown locus heterogeneity with a TSC gene mapped to chromosome 9q34 and a second, recently identified on 16p13.3. We have analysed DNA markers in eight hamartomas and one tumour from TSC patients and found allele loss on 16p13.3 in three angiomyolipomas, one cardiac rhabdomyoma, one cortical tuber and one giant cell astrocytoma. We suggest that the TSC gene on 16p13.3 functions like a tumour suppressor gene, in accordance with Knudsen's hypothesis.
      Cover page: Loss of heterozygosity on chromosome 16p13.3 in hamartomas from tuberous sclerosis patientsCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2005)

      In the pathogenic bacterium Chlamydia trachomatis, a transcriptional repressor, HrcA, regulates the major heat shock operons, dnaK and groE. Cellular stress causes a transient increase in transcription of these heat shock operons through relief of HrcA-mediated repression, but the pathway leading to derepression is unclear. Elevated temperature alone is not sufficient, and it is hypothesized that additional chlamydial factors play a role. We used DNA affinity chromatography to purify proteins that interact with HrcA in vivo and identified a higher-order complex consisting of HrcA, GroEL, and GroES. This endogenous HrcA complex migrated differently than recombinant HrcA, but the complex could be disrupted, releasing native HrcA that resembled recombinant HrcA. In in vitro assays, GroEL increased the ability of HrcA to bind to the CIRCE operator and to repress transcription. Other chlamydial heat shock proteins, including the two additional GroEL paralogs present in all chlamydial species, did not modulate HrcA activity.

        Cover page: Chlamydial GroEL autoregulates its own expression through direct interactions with the HrcA repressor protein.
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        • Peer Reviewed
        UC Berkeley Previously Published Works (2015)
        In budding yeast, over 60 proteins functioning in at least five modules are recruited to endocytic sites with predictable order and timing. However, how sites of clathrin-mediated endocytosis are initiated and stabilized is not well understood. Here, the casein kinase 1 (CK1) Hrr25 is shown to be an endocytic protein and to be among the earliest proteins to appear at endocytic sites. Hrr25 absence or overexpression decreases or increases the rate of endocytic site initiation, respectively. Ede1, an early endocytic Eps15-like protein important for endocytic initiation, is an Hrr25 target and is required for Hrr25 recruitment to endocytic sites. Hrr25 phosphorylation of Ede1 is required for Hrr25-Ede1 interaction and promotes efficient initiation of endocytic sites. These observations indicate that Hrr25 kinase and Ede1 cooperate to initiate and stabilize endocytic sites. Analysis of the mammalian homologs CK1δ/ε suggests a conserved role for these protein kinases in endocytic site initiation and stabilization.
          Cover page: Casein Kinase 1 Promotes Initiation of Clathrin-Mediated Endocytosis
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          • Peer Reviewed
          UC San Diego Previously Published Works (2006)

          We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during import, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner.

            Cover page: Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2016)
            Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.
              Cover page: Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeastCreative Commons 'BY' version 4.0 license
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              • Peer Reviewed
              UC San Francisco Previously Published Works (2013)
              Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress.
                Cover page: Sequential primed kinases create a damage-responsive phosphodegron on Eco1
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2013)
                Dynamic interactions between membrane-bound organelles and the microtubule cytoskeleton are crucial to establish, maintain, and remodel the internal organization of cells throughout the cell cycle. However, the molecular nature of these interactions remains poorly understood. We performed a biochemical screen for microtubule-membrane linkers and identified REEP4, a previously uncharacterized endoplasmic reticulum (ER) protein. Depletion of REEP4 and the closely related REEP3 from HeLa cells causes defects in cell division and a proliferation of intranuclear membranes derived from the nuclear envelope. This phenotype originates in mitosis, when ER membranes accumulate on metaphase chromosomes. Microtubule binding and mitotic ER clearance from chromosomes both depend on a short, positively charged amino acid sequence connecting the two hydrophobic domains of REEP4. Our results show that REEP3/4 function redundantly to clear the ER from metaphase chromatin, thereby ensuring correct progression through mitosis and proper nuclear envelope architecture.
                  Cover page: REEP3/4 Ensure Endoplasmic Reticulum Clearance from Metaphase Chromatin and Proper Nuclear Envelope Architecture
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                  • Peer Reviewed
                  UC Irvine Previously Published Works (2018)
                  Recent technological advances led to the discovery of hundreds to thousands of peptides and small proteins (microproteins) encoded by small open reading frames (smORFs). Characterization of new microproteins demonstrates their role in fundamental biological processes and highlights the value in discovering and characterizing more microproteins. The elucidation of microprotein-protein interactions (MPIs) is useful for determining the biochemical and cellular roles of microproteins. In this study, we characterize the protein interaction partners of mitochondrial elongation factor 1 microprotein (MIEF1-MP) using a proximity labeling strategy that relies on APEX2. MIEF1-MP localizes to the mitochondrial matrix where it interacts with the mitochondrial ribosome (mitoribosome). Functional studies demonstrate that MIEF1-MP regulates mitochondrial translation via its binding to the mitoribosome. Loss of MIEF1-MP decreases the mitochondrial translation rate, while an elevated level of MIEF1-MP increases the translation rate. The identification of MIEF1-MP reveals a new gene involved in this process.
                    Cover page: MIEF1 Microprotein Regulates Mitochondrial Translation.
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                    • Peer Reviewed
                    UC San Diego Previously Published Works (2024)
                    Mass spectrometry-based methods can provide a global expression profile and structural readout of proteins in complex systems. Preserving the in vivo conformation of proteins in their innate state is challenging during proteomic experiments. Here, we introduce a whole animal in vivo protein footprinting method using perfusion of reagents to add dimethyl labels to exposed lysine residues on intact proteins which provides information about protein conformation. When this approach is used to measure dynamic structural changes during Alzheimers disease (AD) progression in a mouse model, we detect 433 proteins that undergo structural changes attributed to AD, independent of aging, across 7 tissues. We identify structural changes of co-expressed proteins and link the communities of these proteins to their biological functions. Our findings show that structural alterations of proteins precede changes in expression, thereby demonstrating the value of in vivo protein conformation measurement. Our method represents a strategy for untangling mechanisms of proteostasis dysfunction caused by protein misfolding. In vivo whole-animal footprinting should have broad applicability for discovering conformational changes in systemic diseases and for the design of therapeutic interventions.
                      Cover page: Using in vivo intact structure for system-wide quantitative analysis of changes in proteins.
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