Daniel E Goldberg

Washington University in St. Louis, San Luis, Missouri, United States

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Publications (139)1047.5 Total impact

  • Daniel E Goldberg
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    ABSTRACT: Export of effector proteins is crucial for the virulence program of the malaria parasite Plasmodium falciparum. A crystal structure of the P. falciparum processing enzyme essential for protein export reveals noncanonical aspartic protease features and provides an avenue for antimalarial drug development.
    No preview · Article · Sep 2015 · Nature Structural & Molecular Biology
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    Paul A Sigala · Jan R Crowley · Jeffrey P Henderson · Daniel E Goldberg
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    ABSTRACT: Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.
    Full-text · Article · Jul 2015 · eLife Sciences
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    ABSTRACT: Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease β-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310μM and 0.099μM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Mar 2015 · Bioorganic & medicinal chemistry
  • Natalie J Spillman · Josh R Beck · Daniel E Goldberg
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    ABSTRACT: Phylum Apicomplexa comprises a large group of obligate intracellular parasites of high medical and veterinary importance. These organisms succeed intracellularly by effecting remarkable changes in a broad range of diverse host cells. The transformation of the host erythrocyte is particularly striking in the case of the malaria parasite Plasmodium falciparum. P. falciparum exports hundreds of proteins that mediate a complex cellular renovation marked by changes in the permeability, rigidity, and cytoadherence properties of the host erythrocyte. The past decade has seen enormous progress in understanding the identity and function of these exported effectors, as well as the mechanisms by which they are trafficked into the host cell. Here we review these advances, place them in the context of host manipulation by related apicomplexans, and propose key directions for future research. Expected final online publication date for the Annual Review of Biochemistry Volume 84 is June 02, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
    No preview · Article · Jan 2015 · Annual Review of Biochemistry
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    ABSTRACT: Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knockout parasite lines, lacking δ-aminolevulinic acid synthase (ALAS) and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 13C4-5-aminolevulinic acid (5-ALA), and this assay confirmed that de novo heme synthesis was ablated in FC knockout parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.
    No preview · Article · Oct 2014 · Journal of Biological Chemistry
  • Jeremy P Mallari · Anna Oksman · Barbara Vaupel · Daniel E Goldberg
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    ABSTRACT: The universally conserved kinase-associated endopeptidase 1 (Kae1) protein family has established roles in N6-threonylcarbamoyl adenosine tRNA modification, transcriptional regulation, and telomere homeostasis. These functions are performed in complex with a conserved core of protein binding partners. Herein we describe the localization, essentiality, and protein-protein interactions for Kae1 in the human malaria parasite Plasmodium falciparum. We found that the parasite expresses one Kae1 protein in the cytoplasm and a second protein in the apicoplast, a chloroplast remnant organelle involved in fatty acid, heme, and isoprenoid biosynthesis. To analyze the protein interaction networks for both Kae1 pathways, we developed a new proteomic cross-validation approach. This strategy compares immunoprecipitation-MS data sets across different cellular compartments to enrich for biologically relevant protein interactions. Our results show that cytoplasmic Kae1 forms a complex with Bud32 and Cgi121 as in other organisms, whereas apicoplast Kae1 makes novel interactions with multiple proteins in the apicoplast. Quantitative RT-PCR and immunoprecipitation studies indicate that apicoplast Kae1 and its partners interact specifically with the apicoplast ribosomes and with proteins involved in ribosome function. Together, these data indicate an expanded, apicoplast-specific role for Kae1 in the parasite.
    No preview · Article · Sep 2014 · Journal of Biological Chemistry
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    Josh R Beck · Vasant Muralidharan · Anna Oksman · Daniel E Goldberg
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    ABSTRACT: To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.
    Full-text · Article · Jul 2014 · Nature
  • Paul A Sigala · Daniel E Goldberg
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    ABSTRACT: For over a century, heme metabolism has been recognized to play a central role during intraerythrocytic infection by Plasmodium parasites, the causative agent of malaria. Parasites liberate vast quantities of potentially cytotoxic heme as a by-product of hemoglobin catabolism within the digestive vacuole, where heme is predominantly sequestered as inert crystalline hemozoin. Plasmodium spp. also utilize heme as a metabolic cofactor. Despite access to abundant host-derived heme, parasites paradoxically maintain a biosynthetic pathway. This pathway has been assumed to produce the heme incorporated into mitochondrial cytochromes that support electron transport. In this review, we assess our current understanding of the love-hate relationship between Plasmodium parasites and heme, we discuss recent studies that clarify several long-standing riddles about heme production and utilization by parasites, and we consider remaining challenges and opportunities for understanding and targeting heme metabolism within parasites. Expected final online publication date for the Annual Review of Microbiology Volume 68 is September 08, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
    No preview · Article · Jun 2014 · Annual Review of Microbiology
  • Ilaria Russo · Daniel E. Goldberg

    No preview · Chapter · Dec 2013
  • Colin Berry · Daniel E. Goldberg

    No preview · Chapter · Dec 2013
  • Colin Berry · Daniel E. Goldberg

    No preview · Chapter · Dec 2013
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    ABSTRACT: Given the threat of drug resistance, there is an acute need for new classes of antimalarial agents that act via a unique mechanism of action relative to currently used drugs. We have identified a set of druglike compounds within the Tres Cantos Anti-Malarial Set (TCAMS) which likely act via inhibition of a Plasmodium aspartic protease. Structure-activity relationship analysis and optimization of these aminohydantoins demonstrate that these compounds are potent nanomolar inhibitors of the Plasmodium aspartic proteases PM-II and PM-IV and likely one or more other Plasmodium aspartic proteases. Incorporation of a bulky group, such as a cyclohexyl group, on the aminohydantion N-3 position gives enhanced antimalarial potency while reducing inhibition of human aspartic proteases such as BACE. We have identified compound 8p (CWHM-117) as a promising lead for optimization as an antimalarial drug with a low molecular weight, modest lipophilicity, oral bioavailability, and in vivo antimalarial activity in mice.
    Full-text · Article · Dec 2013 · ACS Medicinal Chemistry Letters
  • Daniel E. Goldberg · Min Zhang · Victor Nussenzweig
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    ABSTRACT: eIF2α kinases are stress sensors that respond to external stimuli by phosphorylating the eukaryotic initiation factor eIF2α. This response downregulates cellular protein synthesis but induces the translation of select mRNAs to allow stress survival. In Plasmodium, there are three eIF2α kinases: PfeIK1 responds to amino acid deprivation, while the other two are involved in controlling parasite development. Of the latter kinases, PfPK4 is essential for intraerythrocytic survival, while PfeIK2 controls salivary gland sporozoite latency. The study of parasite eIF2α kinases is broadening the present view of the role of these kinases in cellular and organismal function.
    No preview · Chapter · Oct 2013
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    Vasant Muralidharan · Daniel E Goldberg

    Full-text · Article · Aug 2013 · PLoS Pathogens
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    Daniel E Goldberg

    Preview · Article · Mar 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Malaria parasites multiply in human erythrocytes through schizogony, a process characterised by nuclear divisions in the absence of cytokinesis, leading to the formation of a multinucleated schizont from which individual daughter cells are subsequently generated. Here, we provide evidence that parasites lines lacking Pfcrk-5, an atypical cyclin-dependent kinase, display a reduced parasitemia growth rate linked to a decrease in the number of daughter nuclei produced by each schizont. We show that in vitro activity of recombinant Pfcrk-5 is indeed cyclin-dependent, and that the enzyme localises to the nuclear periphery. Thus, Pfcrk-5 is part of a regulatory pathway that mediates the proliferation rate of Plasmodium falciparum through the control of nuclear divisions during schizogony.
    Full-text · Article · Mar 2013
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    ABSTRACT: One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes.
    Preview · Article · Dec 2012 · Nature Communications
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    Daniel E Goldberg
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    ABSTRACT: Intraerythrocytic malaria parasites send hundreds of effector proteins into the host cell. Diverse modes of export have been proposed for different proteins. In this issue, Grüring et al. (2012) present findings that bring the models together.
    Preview · Article · Nov 2012 · Cell host & microbe
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    ABSTRACT: The human malaria parasite Plasmodium falciparum is auxotrophic for most amino acids. Its amino acid needs are met largely through the degradation of host erythrocyte hemoglobin; however the parasite must acquire isoleucine exogenously, because this amino acid is not present in adult human hemoglobin. We report that when isoleucine is withdrawn from the culture medium of intraerythrocytic P. falciparum, the parasite slows its metabolism and progresses through its developmental cycle at a reduced rate. Isoleucine-starved parasites remain viable for 72 h and resume rapid growth upon resupplementation. Protein degradation during starvation is important for maintenance of this hibernatory state. Microarray analysis of starved parasites revealed a 60% decrease in the rate of progression through the normal transcriptional program but no other apparent stress response. Plasmodium parasites do not possess a TOR nutrient-sensing pathway and have only a rudimentary amino acid starvation-sensing eukaryotic initiation factor 2α (eIF2α) stress response. Isoleucine deprivation results in GCN2-mediated phosphorylation of eIF2α, but kinase-knockout clones still are able to hibernate and recover, indicating that this pathway does not directly promote survival during isoleucine starvation. We conclude that P. falciparum, in the absence of canonical eukaryotic nutrient stress-response pathways, can cope with an inconsistent bloodstream amino acid supply by hibernating and waiting for more nutrient to be provided.
    Full-text · Article · Oct 2012 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Malaria parasites generate vast quantities of heme during blood-stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygense (HO)-like protein from P. falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme-degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with the lack of a heme-coordinating His residue in PfHO and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood-stage infection.
    No preview · Article · Sep 2012 · Journal of Biological Chemistry

Publication Stats

9k Citations
1,047.50 Total Impact Points

Institutions

  • 1992-2015
    • Washington University in St. Louis
      • • Department of Medicine
      • • Department of Molecular Microbiology
      San Luis, Missouri, United States
  • 1995-2014
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2005-2006
    • University of California, San Francisco
      • Department of Medicine
      San Francisco, California, United States
  • 1996-1997
    • Albert Einstein College of Medicine
      • Department of Physiology & Biophysics
      New York City, NY, United States
  • 1990-1991
    • The Rockefeller University
      New York City, New York, United States