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Open Access Publications from the University of California

Scholarly Works (36 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2020)
Apicomplexans form a large phylum of parasitic protozoa, including the genera Plasmodium, Toxoplasma, and Cryptosporidium, the causative agents of malaria, toxoplasmosis, and cryptosporidiosis, respectively. They cause diseases not only in humans but also in animals, with dramatic consequences in agriculture. Most apicomplexans are vacuole-dwelling and obligate intracellular parasites; as they invade the host cell, they become encased in a parasitophorous vacuole (PV) derived from the host cellular membrane. This creates a parasite-host interface that acts as a protective barrier but also constitutes an obstacle through which the pathogen must import nutrients, eliminate wastes, and eventually break free upon egress. Completion of the parasitic life cycle requires intense remodeling of the infected host cell. Host cell subversion is mediated by a subset of essential effector parasitic proteins and virulence factors actively trafficked across the PV membrane. In the malaria parasite Plasmodium, a unique and highly specialized ATP-driven vacuolar secretion system, the Plasmodium translocon of exported proteins (PTEX), transports effector proteins across the vacuolar membrane. Its core is composed of the three essential proteins EXP2, PTEX150, and HSP101, and is supplemented by the two auxiliary proteins TRX2 and PTEX88. Many but not all secreted malarial effector proteins contain a vacuolar trafficking signal or Plasmodium export element (PEXEL) that requires processing by an endoplasmic reticulum protease, plasmepsin V, for proper export. Because vacuolar parasitic protein export is essential to parasite survival and virulence, this pathway is a promising target for the development of novel antimalarial therapeutics. This review summarizes the current state of structural and mechanistic knowledge on the Plasmodium parasitic vacuolar secretion and effector trafficking pathway, describing its most salient features and discussing the existing differences and commonalities with the vacuolar effector translocation MYR machinery recently described in Toxoplasma and other apicomplexans of significance to medical and veterinary sciences.
    Cover page: Crossing the Vacuolar Rubicon: Structural Insights into Effector Protein Trafficking in Apicomplexan Parasites
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2022)
      Cover page: Moving Lipids, by the Numbers
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2021)
      Eukaryotic cells are characterized by their exquisite compartmentalization resulting from a cornucopia of membrane-bound organelles. Each of these compartments hosts a flurry of biochemical reactions and supports biological functions such as genome storage, membrane protein and lipid biosynthesis/degradation and ATP synthesis, all essential to cellular life. Acting as hubs for the transfer of matter and signals between organelles and throughout the cell, membrane contacts sites (MCSs), sites of close apposition between membranes from different organelles, are essential to cellular homeostasis. One of the now well-acknowledged function of MCSs involves the non-vesicular trafficking of lipids; its characterization answered one long-standing question of eukaryotic cell biology revealing how some organelles receive and distribute their membrane lipids in absence of vesicular trafficking. The endoplasmic reticulum (ER) in synergy with the mitochondria, stands as the nexus for the biosynthesis and distribution of phospholipids (PLs) throughout the cell by contacting nearly all other organelle types. MCSs create and maintain lipid fluxes and gradients essential to the functional asymmetry and polarity of biological membranes throughout the cell. Membrane apposition is mediated by proteinaceous tethers some of which function as lipid transfer proteins (LTPs). We summarize here the current state of mechanistic knowledge of some of the major classes of LTPs and tethers based on the available atomic to near-atomic resolution structures of several "model" MCSs from yeast but also in Metazoans; we describe different models of lipid transfer at MCSs and analyze the determinants of their specificity and directionality. Each of these systems illustrate fundamental principles and mechanisms for the non-vesicular exchange of lipids between eukaryotic membrane-bound organelles essential to a wide range of cellular processes such as at PL biosynthesis and distribution, lipid storage, autophagy and organelle biogenesis.
        Cover page: Mechanisms of Non-Vesicular Exchange of Lipids at Membrane Contact Sites: Of Shuttles, Tunnels and, Funnels
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2001)
        The purification and crystallization of the stoichiometric complex of human RXRalpha ligand-binding domain (hRXRalpha LBD) bound to its natural ligand 9-cis retinoic acid (9-cisRA) are described. A three-step purification yields a pure and homogeneous complex. Based on the crystallization conditions of several other nuclear receptors, an exhaustive crystallization screening using carboxylic acids as precipitating agents was performed in association with the use of polyhydric alcohols acting as cosmotropic solutes. Crystals of the hRXRalpha LBD-9-cisRA complex grew in a tripartite mixture containing sodium formate, glycerol and propane-1,2-diol. Micro- and macroseeding were necessary to improve both the size and the quality of crystals in order to make them suitable for structure determination.
          Cover page: Purification and crystallization of the human RXRα ligand‐binding domain–9‐cisRA complex
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2010)
          The structure of the protein-translocating channel SecYEβ from Pyrococcus furiosus at 3.1-Å resolution suggests a mechanism for chaperoning transmembrane regions of a protein substrate during its lateral delivery into the lipid bilayer. Cytoplasmic segments of SecY orient the C-terminal α-helical region of another molecule, suggesting a general binding mode and a promiscuous guiding surface capable of accommodating diverse nascent chains at the exit of the ribosomal tunnel. To accommodate this putative nascent chain mimic, the cytoplasmic vestibule widens, and a lateral exit portal is opened throughout its entire length for partition of transmembrane helical segments to the lipid bilayer. In this primed channel, the central plug still occludes the pore while the lateral gate is opened, enabling topological arbitration during early protein insertion. In vivo, a 15 amino acid truncation of the cytoplasmic C-terminal helix of SecY fails to rescue a secY-deficient strain, supporting the essential role of this helix as suggested from the structure.
            Cover page: Lateral opening of a translocon upon entry of protein suggests the mechanism of insertion into membranes
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2002)
            The nuclear receptor RXR is an obligate partner in many signal transduction pathways. We report the high-resolution structures of two complexes of the human RXRalpha ligand-binding domain specifically bound to two different and chemically unrelated agonist compounds: docosa hexaenoic acid, a natural derivative of eicosanoic acid, present in mammalian cells and recently identified as a potential endogenous RXR ligand in the mouse brain, and the synthetic ligand BMS 649. In both structures the RXR-ligand-binding domain forms homodimers and exhibits the active conformation previously observed with 9-cis-RA. Analysis of the differences in ligand-protein contacts (predominantly van der Waals forces) and binding cavity geometries and volumes for the several agonist-bound RXR structures clarifies the structural features important for ligand recognition. The L-shaped ligand-binding pocket adapts to the diverse ligands, especially at the level of residue N306, which might thus constitute a new target for drug-design. Despite its highest affinity 9-cis-RA displays the lowest number of ligand-protein contacts. These structural results support the idea that docosa hexaenoic acid and related fatty acids could be natural agonists of RXRs and question the real nature of the endogenous ligand(s) in mammalian cells.
              Cover page: Molecular Recognition of Agonist Ligands by RXRs
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2019)
              Membrane contact sites between the endoplasmic reticulum (ER) and mitochondria function as a central hub for the exchange of phospholipids and calcium. The yeast Endoplasmic Reticulum-Mitochondrion Encounter Structure (ERMES) complex is composed of five subunits that tether the ER and mitochondria. Three ERMES subunits (i.e., Mdm12, Mmm1, and Mdm34) contain the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. The SMP domain belongs to the tubular lipid-binding protein (TULIP) superfamily, which consists of ubiquitous lipid scavenging and transfer proteins. Herein, we describe the methods for expression and purification of recombinant Mdm12, a bona fide SMP-containing protein, together with the subsequent identification of its bound phospholipids by high-performance thin-layer chromatography (HPTLC) and the characterization of its lipid exchange and transfer functions using lipid displacement and liposome flotation in vitro assays with liposomes as model biological membranes. These methods can be applied to the study and characterization of novel lipid-binding and lipid-transfer proteins.
                Cover page: Determining the Lipid-Binding Specificity of SMP Domains: An ERMES Subunit as a Case Study
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2015)
                Survival of the malaria parasite Plasmodium falciparum when it infects red blood cells depends upon its ability to export hundreds of its proteins beyond an encasing vacuole. Protein export is mediated by a parasite-derived protein complex, the Plasmodium translocon of exported proteins (PTEX), and requires unfolding of the different cargos prior to their translocation across the vacuolar membrane. Unfolding is performed by the AAA+protein unfoldase HSP101/ClpB2 and the thioredoxin-2 enzyme (TRX2). Protein trafficking is dramatically impaired in parasites with defective HSP101 or lacking TRX2. These two PTEX subunits drive export and are targets for the design of a novel class of antimalarials: protein export inhibitors. To rationalize inhibitor design, we solved the crystal structure of Pfal-TRX2 at 2.2-Å resolution. Within the asymmetric unit, the three different copies of this protein disulfide reductase sample its two redox catalytic states. Size exclusion chromatography and small-angle X-ray scattering (SAXS) analyses demonstrate that Pfal-TRX2 is monomeric in solution. A non-conserved N-terminal extension precedes the canonical thioredoxin-fold; although it is not observed in our structure, our solution analysis suggests it is flexible in contrast to Plasmodium thioredoxin-1. This represents a first step towards the reconstitution of the entire PTEX for mechanistic and structural studies.
                  Cover page: Crystal structure and solution characterization of the thioredoxin-2 from Plasmodium falciparum, a constituent of an essential parasitic protein export complex
                  • Thesis
                  • Peer Reviewed
                  UCLA Electronic Theses and Dissertations (2015)

                  This dissertation describes a comprehensive set of experiments aimed at understanding two distinct biological phenomena that are essential to the cell—1) Interorganelle communication, and 2) regulation of protein unfolding and degradation.

                  The compartmentalization of cells permits the segregation and regulation of diverse and specialized biochemical reactions. The tremendous benefits of intracellular compartmentalization also come at a price; to function harmoniously, cells must coordinate the exchange of biological information between compartments. Numerous mechanisms have evolved to facilitate these exchanges. One that has not been well appreciated until the last few years is the transmission of signals between organelles at regions where the organelles are closely apposed, often called membrane contact sites (MCSs). Emerging evidence highlight essential roles for interorganelle tethering complexes in the formation and maintenance of MCS. These molecular tethers are widespread and function primarily by bridging two organelles, including the endoplasmic reticulum (ER) and mitochondria.

                  Chapter 2 discusses the structural and biochemical characterization of the multi-subunit interorganelle tethering complex ERMES (endoplasmic reticulum-mitochondrial encounter structure), which bridges the ER and mitochondria in the yeast, Saccharomyces cerevisiae. To our knowledge, this is the first structure of a multi-subunit tether known to date. Our findings highlight novel roles for the conserved SMP domain (synaptotagmin-like mitochondrial and lipid-binding protein)—present in three ERMES subunits—in ERMES assembly and phospholipid binding, and suggest a structure-based mechanism for the facilitated transport of phospholipids between the ER and mitochondria.

                  Intracellular proteolysis is regulated by a wide array of molecular systems consisting of molecular chaperones and proteases. These surveillance systems ensure that misfolded proteins and short-lived regulatory proteins are targeted for degradation. The Clp family of chaperones and proteases play an important role in protein homeostasis in the cell. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Several members of the Clp family were recently identified in Plasmodium falciparum, the etiologic agent of malaria. Chapters 3 and 4 detail the structures of three members of the Plasmodium Clp family—the AAA+ chaperones ClpB1 and ClpB2, and the adaptor protein ClpS. These structures provide novel insights into the role of the elusive Plasmodium Clp protease machinery in protein unfolding and degradation, and open new venues for the design of novel anti-malarial drugs aimed at disrupting parasite-specific, protein quality control pathways.

                    Cover page: Structural and Biochemical Characterization of Eukaryotic Protein Complexes Involved in Interorganelle Tethering and Protein Quality Control
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2015)
                    Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains-a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs.
                      Cover page: Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design
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