Daniel E Goldberg

University of Georgia, Атина, Georgia, United States

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Publications (112)849.91 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The universally conserved kinase-associated endopeptidase 1 (Kae1) protein family has established roles in t6A (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 find that the parasite expresses one Kae1 protein in the cytoplasm and a second 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 IP-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, while apicoplast Kae1 makes novel interactions with multiple proteins in the apicoplast. qRT-PCR and immunoprecipitation studies indicate 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.
    Journal of Biological Chemistry 09/2014; · 4.65 Impact Factor
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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.
    Nature 07/2014; · 38.60 Impact Factor
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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. KEYWORDS: Malaria, antimalarial, aminohydantoin, medicinal chemistry, aspartic protease inhibitors M alaria is a devastating mosquito-borne infectious disease caused by a parasite of the genus Plasmodium, placing over one billion people at high risk for infection. According to the World Health Organization, there were an estimated 225 million cases of malaria in 2010 with 610,000−971,000 deaths. 1 Especially hard hit is sub-Saharan Africa, where 80% of the deaths occur, mostly in children under the age of 5 years old. Although there are a number of drugs used to treat the disease, resistance to most of these drugs is widespread. 2 The introduction of artemisinin and artemisinin combination therapies (ACTs) in 2005 has begun to reverse the trend. While this is a good sign, there have been reports of resistance to artemisinin in Southeast Asia. 3 As a consequence, there is an urgent push for developing antimalarial therapies targeting novel modes of action. Drug discovery efforts in this area have been recently reviewed. 4−6
    ACS Medicinal Chemistry Letters 12/2013; 5(1):89-93. · 3.31 Impact Factor
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    Vasant Muralidharan, Daniel E Goldberg
    PLoS Pathogens 08/2013; 9(8):e1003488. · 8.14 Impact Factor
  • Daniel E Goldberg
    Proceedings of the National Academy of Sciences 03/2013; · 9.81 Impact Factor
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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.
    KInome. 03/2013; 1:4–16.
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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.
    Nature Communications 12/2012; 3:1310. · 10.74 Impact Factor
  • 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.
    Cell host & microbe 11/2012; 12(5):609-10. · 13.02 Impact Factor
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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.
    Proceedings of the National Academy of Sciences 10/2012; · 9.81 Impact Factor
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    Daniel E Goldberg, Robert F Siliciano, William R Jacobs
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    ABSTRACT: Although caused by vastly different pathogens, the world's three most serious infectious diseases, tuberculosis, malaria, and HIV-1 infection, share the common problem of drug resistance. The pace of drug development has been very slow for tuberculosis and malaria and rapid for HIV-1. But for each disease, resistance to most drugs has appeared quickly after the introduction of the drug. Learning how to manage and prevent resistance is a major medical challenge that requires an understanding of the evolutionary dynamics of each pathogen. This Review summarizes the similarities and differences in the evolution of drug resistance for these three pathogens.
    Cell 03/2012; 148(6):1271-83. · 31.96 Impact Factor
  • Marvin J Meyers, Daniel E Goldberg
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    ABSTRACT: Plasmepsins are the aspartic proteases of Plasmodium that play key roles in the survival of the parasite in its host. The plasmepsins of the digestive vacuole play an important role in hemoglobin degradation, providing the parasite with a vital source of nutrients. Recently, plasmepsin V has been shown to be an essential protease, processing hundreds of parasite proteins for export into the host erythrocyte. The functions of the remaining plasmepsins have yet to be discovered. Over the past decade, much effort has been placed towards developing plasmepsin inhibitors as antimalarial agents, particularly targeting the digestive vacuole. This review will highlight some of the recent work in this field with a particular focus on target druggability and strategies for identifying plasmepsins inhibitors as effective antimalarial drugs. Given recent advances in understanding the fundamental roles of the various plasmepsins, it is likely that the most effective antimalarial plasmepsin targets will be the non-digestive vacuole plasmepsins.
    Current topics in medicinal chemistry 03/2012; 12(5):445-55. · 4.47 Impact Factor
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    ABSTRACT: The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC(50) values in the low micromolar range (2-48μM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1' subsite residues whereas the acetophenone moiety was in contact with S1-S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ⩽3μM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC(50) of 160nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.
    Bioorganic & medicinal chemistry letters 10/2011; 22(2):1282-6. · 2.65 Impact Factor
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    ABSTRACT: One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.
    Proceedings of the National Academy of Sciences 02/2011; 108(11):4411-6. · 9.81 Impact Factor
  • Trends in Parasitology 01/2011; 27(1):1-2. · 5.51 Impact Factor
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    ABSTRACT: Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucine-resistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum. Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development.
    Proceedings of the National Academy of Sciences 01/2011; 108(4):1627-32. · 9.81 Impact Factor
  • Daniel E Goldberg, Alan F Cowman
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    ABSTRACT: Malaria parasites live within erythrocytes in the host bloodstream and induce crucial changes to these cells. By so doing, they can obtain the nutrients that they require for growth and can effect the evasion and perturbation of host defences. In order to accomplish this extensive host cell remodelling, the intracellular parasite exports hundreds of proteins to commander the erythrocyte for its own purposes. An export motif, a processing enzyme that specifies protein targeting and a translocon that mediates the export of proteins from the parasite into the host erythrocyte have been identified. However, important questions remain regarding the secretory pathway and the function of the translocon. In addition, this export pathway provides potentially useful targets for the development of inhibitors to interfere with functions that are vital for the virulence and survival programmes of the parasite.
    Nature Reviews Microbiology 09/2010; 8(9):617-21. · 22.49 Impact Factor
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    ABSTRACT: During their intraerythrocytic development, malaria parasites export hundreds of proteins to remodel their host cell. Nutrient acquisition, cytoadherence and antigenic variation are among the key virulence functions effected by this erythrocyte takeover. Proteins destined for export are synthesized in the endoplasmic reticulum (ER) and cleaved at a conserved (PEXEL) motif, which allows translocation into the host cell via an ATP-driven translocon called the PTEX complex. We report that plasmepsin V, an ER aspartic protease with distant homology to the mammalian processing enzyme BACE, recognizes the PEXEL motif and cleaves it at the correct site. This enzyme is essential for parasite viability and ER residence is essential for its function. We propose that plasmepsin V is the PEXEL protease and is an attractive enzyme for antimalarial drug development.
    Nature 02/2010; 463(7281):632-6. · 38.60 Impact Factor
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    Ilaria Russo, Anna Oksman, Daniel E Goldberg
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    ABSTRACT: The Plasmodium falciparum genome encodes a single calpain. By generating P. falciparum clones expressing C-terminally tagged calpain, we localized this protein to the nucleolus. Pf_calpain possesses an unusual and long N-terminal domain in which we identified three subregions that are highly conserved among Plasmodium species. Two have putative targeting signals: a myristoylation motif and a nuclear localization sequence. We assessed their functionality. Our data show that the nuclear localization sequence is an active nuclear import motif that contains an embedded signal conferring nucleolar localization on various chimeras. The N-terminus is myristoylated at Gly2 and palmitoylated at Cys3 and Cys22. Palmitoylation status has an important role in dictating P. falciparum calpain localization. The targeting signals function in mammalian cells as well as in the parasite. P. falciparum calpain is a unique nucleolar protein with an interesting mechanism of targeting.
    Molecular Microbiology 03/2009; 72(1):229-45. · 5.03 Impact Factor
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    ABSTRACT: Post-transcriptional control of gene expression is suspected to play an important role in malaria parasites. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), which results in the selective translation of mRNAs encoding stress-response proteins. The impact of starvation on the phosphorylation state of PfeIF2alpha was examined. Bioinformatic methods were used to identify plasmodial eIF2alpha kinases. The activity of one of these, PfeIK1, was investigated using recombinant protein with non-physiological substrates and recombinant PfeIF2alpha. Reverse genetic techniques were used to disrupt the pfeik1 gene. The data demonstrate that the Plasmodium falciparum eIF2alpha orthologue is phosphorylated in response to starvation, and provide bioinformatic evidence for the presence of three eIF2alpha kinases in P. falciparum, only one of which (PfPK4) had been described previously. Evidence is provided that one of the novel eIF2alpha kinases, PfeIK1, is able to phosphorylate the P. falciparum eIF2alpha orthologue in vitro. PfeIK1 is not required for asexual or sexual development of the parasite, as shown by the ability of pfeik1- parasites to develop into sporozoites. However, eIF2alpha phosphorylation in response to starvation is abolished in pfeik1- asexual parasites This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain but distinct regulatory domains, is conserved in P. falciparum.
    Malaria Journal 02/2009; 8:99. · 3.49 Impact Factor
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    Ilaria Russo, Anna Oksman, Barbara Vaupel, Daniel E Goldberg
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    ABSTRACT: Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.
    Proceedings of the National Academy of Sciences 02/2009; 106(5):1554-9. · 9.81 Impact Factor

Publication Stats

4k Citations
849.91 Total Impact Points

Institutions

  • 2013
    • University of Georgia
      Атина, Georgia, United States
  • 1995–2013
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2012
    • Saint Louis University
      • Department of Molecular Microbiology & Immunology
      Saint Louis, MI, United States
  • 1993–2008
    • Washington University in St. Louis
      • • Department of Molecular Microbiology
      • • Department of Biology
      • • Department of Medicine
      San Luis, Missouri, United States
  • 2004–2006
    • University of California, San Francisco
      • Division of Hospital Medicine
      San Francisco, CA, United States
  • 2002
    • Mahidol University
      • Faculty of Science
      Bangkok, Bangkok, Thailand
  • 1999
    • University of California, Davis
      • Department of Chemistry
      Davis, CA, United States
  • 1998
    • National Cancer Institute (USA)
      Maryland, United States
    • Johns Hopkins Medicine
      • Department of Molecular Microbiology and Immunology
      Baltimore, MD, United States
  • 1993–1997
    • University of Washington Seattle
      • Department of Medicine
      Seattle, WA, United States