DrugBank 6.0: the DrugBank Knowledgebase for 2024.

First released in 2006, DrugBank (https://go.drugbank.com) has grown to become the 'gold standard' knowledge resource for drug, drug-target and related pharmaceutical information. DrugBank is widely used across many diverse biomedical research and clinical applications, and averages more than 30 million views/year. Since its last update in 2018, we have been actively enhancing the quantity and quality of the drug data in this knowledgebase. In this latest release (DrugBank 6.0), the number of FDA approved drugs has grown from 2646 to 4563 (a 72% increase), the number of investigational drugs has grown from 3394 to 6231 (a 38% increase), the number of drug-drug interactions increased from 365 984 to 1 413 413 (a 300% increase), and the number of drug-food interactions expanded from 1195 to 2475 (a 200% increase). In addition to this notable expansion in database size, we have added thousands of new, colorful, richly annotated pathways depicting drug mechanisms and drug metabolism. Likewise, existing datasets have been significantly improved and expanded, by adding more information on drug indications, drug-drug interactions, drug-food interactions and many other relevant data types for 11 891 drugs. We have also added experimental and predicted MS/MS spectra, 1D/2D-NMR spectra, CCS (collision cross section), RT (retention time) and RI (retention index) data for 9464 of DrugBank's 11 710 small molecule drugs. These and other improvements should make DrugBank 6.0 even more useful to a much wider research audience ranging from medicinal chemists to metabolomics specialists to pharmacologists.

AAK1-like: A putative pseudokinase with potential roles in cargo uptake in bloodstream form Trypanosoma brucei parasites.

Selection and internalization of cargo via clathrin-mediated endocytosis requires adaptor protein complexes. One complex, AP-2, acts during cargo selection at the plasma membrane. African trypanosomes lack all components of the AP-2 complex, except for a recently identified orthologue of the AP-2-associated protein kinase 1, AAK1. In characterized eukaryotes, AAK1 phosphorylates the µ2 subunit of the AP-2 complex to enhance cargo recognition and uptake into clathrin-coated vesicles. Here, we show that kinetoplastids encode not one, but two AAK1 orthologues: one (AAK1L2) is absent from salivarian trypanosomes, while the other (AAK1L1) lacks important kinase-specific residues in a range of trypanosomes. These AAK1L1 and AAK1L2 novelties reinforce suggestions of functional divergence in endocytic uptake within salivarian trypanosomes. Despite this, we show that AAK1L1 null mutant Trypanosoma brucei, while viable, display slowed proliferation, morphological abnormalities including swelling of the flagellar pocket, and altered cargo uptake. In summary, our data suggest an unconventional role for a putative pseudokinase during endocytosis and/or vesicular trafficking in T. brucei, independent of AP-2.

An endocytic-secretory cycle participates in Toxoplasma gondii in motility.

Apicomplexan parasites invade host cells in an active process involving their ability to move by gliding motility. While the acto-myosin system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions regarding the involvement of other mechanisms in parasite motility. In many eukaryotes, a secretory-endocytic cycle leads to the recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility, and generation of retrograde membrane flow. Here, we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, because inhibition of endocytosis blocks retrograde flow and motility. We demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as labelled phospholipids, nanogold particles (NGPs), antibodies, and Concanavalin A (ConA). Using labelled phospholipids, we observed that the endocytic and secretory pathways of the parasite converge, and endocytosed lipids are subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings, and we propose an additional model, working in parallel to the acto-myosin motor, that reconciles parasite motility with observations in other eukaryotes: an apicomplexan fountain-flow-model for parasite motility.

A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins.

Peroxisomes are membrane-bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence-based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope-tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well-developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease.

Progressive and Biased Divergent Evolution Underpins the Origin and Diversification of Peridinin Dinoflagellate Plastids.

Dinoflagellates are algae of tremendous importance to ecosystems and to public health. The cell biology and genome organization of dinoflagellate species is highly unusual. For example, the plastid genomes of peridinin-containing dinoflagellates encode only a minimal number of genes arranged on small elements termed "minicircles". Previous studies of peridinin plastid genes have found evidence for divergent sequence evolution, including extensive substitutions, novel insertions and deletions, and use of alternative translation initiation codons. Understanding the extent of this divergent evolution has been hampered by the lack of characterized peridinin plastid sequences. We have identified over 300 previously unannotated peridinin plastid mRNAs from published transcriptome projects, vastly increasing the number of sequences available. Using these data, we have produced a well-resolved phylogeny of peridinin plastid lineages, which uncovers several novel relationships within the dinoflagellates. This enables us to define changes to plastid sequences that occurred early in dinoflagellate evolution, and that have contributed to the subsequent diversification of individual dinoflagellate clades. We find that the origin of the peridinin dinoflagellates was specifically accompanied by elevations both in the overall number of substitutions that occurred on plastid sequences, and in the Ka/Ks ratio associated with plastid sequences, consistent with changes in selective pressure. These substitutions, alongside other changes, have accumulated progressively in individual peridinin plastid lineages. Throughout our entire dataset, we identify a persistent bias toward non-synonymous substitutions occurring on sequences encoding photosystem I subunits and stromal regions of peridinin plastid proteins, which may have underpinned the evolution of this unusual organelle.

Tracing the Archaeal Origins of Eukaryotic Membrane-Trafficking System Building Blocks.

In contrast to prokaryotes, eukaryotic cells are characterized by a complex set of internal membrane-bound compartments. A subset of these, and the protein machineries that move material between them, define the membrane-trafficking system (MTS), the emergence of which represents a landmark in eukaryotic evolution. Unlike mitochondria and plastids, MTS organelles have autogenous origins. Much of the MTS machinery is composed of building blocks, including small GTPase, coiled-coil, beta-propeller + alpha-solenoid, and longin domains. Despite the identification of prokaryotic proteins containing these domains, only few represent direct orthologues, leaving the origins and early evolution of the MTS poorly understood. Here, we present an in-depth analysis of MTS building block homologues in the composite genome of Lokiarchaeum, the recently discovered archaeal sister clade of eukaryotes, yielding several key insights. We identify two previously unreported Eukaryotic Signature Proteins; orthologues of the Gtr/Rag family GTPases, involved in target of rapamycin complex signaling, and of the RLC7 dynein component. We could not identify golgin or SNARE (coiled-coil) or beta-propeller + alpha-solenoid orthologues, nor typical MTS domain fusions, suggesting that these either were lost from Lokiarchaeum or emerged later in eukaryotic evolution. Furthermore, our phylogenetic analyses of lokiarchaeal GTPases support a split into Ras-like and Arf-like superfamilies, with different prokaryotic antecedents, before the advent of eukaryotes. While no GTPase activating proteins or exchange factors were identified, we show that Lokiarchaeum encodes numerous roadblock domain proteins and putative longin domain proteins, confirming the latter's origin from Archaea. Altogether, our study provides new insights into the emergence and early evolution of the eukaryotic membrane-trafficking system.

A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum possesses sophisticated systems of protein secretion to modulate host cell invasion and remodeling. In the present study, we provide insights into the function of the AP-1 complex in P. falciparum. We utilized GFP fusion constructs for live cell imaging, as well as fixed parasites in immunofluorescence analysis, to study adaptor protein mu1 (Pfµ1) mediated protein trafficking in P. falciparum. In trophozoites Pfµ1 showed similar dynamic localization to that of several Golgi/ER markers, indicating Golgi/ER localization. Treatment of transgenic parasites with Brefeldin A altered the localization of Golgi-associated Pfµ1, supporting the localization studies. Co-localization studies showed considerable overlap of Pfµ1 with the resident rhoptry proteins, rhoptry associated protein 1 (RAP1) and Cytoadherence linked asexual gene 3.1 (Clag3.1) in schizont stage. Immunoprecipitation experiments with Pfµ1 and PfRAP1 revealed an interaction, which may be mediated through an intermediate transmembrane cargo receptor. A specific role for Pfµ1 in trafficking was suggested by treatment with AlF4, which resulted in a shift to a predominantly ER-associated compartment and consequent decrease in co-localization with the Golgi marker GRASP. Together, these results suggest a role for the AP-1 complex in rhoptry protein trafficking in P. falciparum.

Evolutionary analysis identifies a Golgi pathway and correlates lineage-specific factors with endomembrane organelle emergence in apicomplexans.

The organelle paralogy hypothesis (OPH) aims to explain the evolution of non-endosymbiotically derived organelles. It predicts that lineage-specific pathways or organelles should result when identity-encoding membrane-trafficking components duplicate and co-evolve. Here, we investigate the presence of such lineage-specific membrane-trafficking machinery paralogs in Apicomplexa, a globally important parasitic lineage. We are able to identify 18 paralogs of known membrane-trafficking machinery, in several cases co-incident with the presence of new endomembrane organelles in apicomplexans or their parent lineage, the Alveolata. Moreover, focused analysis of the apicomplexan Arf-like small GTPases (i.e., ArlX3) revealed a specific post-Golgi trafficking pathway. This pathway appears involved in delivery of proteins to micronemes and rhoptries, with knockdown demonstrating reduced invasion capacity. Overall, our data have identified an unforeseen post-Golgi trafficking pathway in apicomplexans and are consistent with the OPH mechanism acting to produce endomembrane pathways or organelles at various evolutionary stages across the alveolate lineage.

Stable endocytic structures navigate the complex pellicle of apicomplexan parasites.

Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

Evolution and Natural History of Membrane Trafficking in Eukaryotes.

The membrane-trafficking system is a defining facet of eukaryotic cells. The best-known organelles and major protein families of this system are largely conserved across the vast diversity of eukaryotes, implying both ancient organization and functional unity. Nonetheless, intriguing variation exists that speaks to the evolutionary forces that have shaped the endomembrane system in eukaryotes and highlights ways in which membrane trafficking in protists differs from that in our well-understood models of mammalian and yeast cells. Both parasites and free-living protists possess specialized trafficking organelles, some lineage specific, others more widely distributed - the evolution and function of these organelles begs exploration. Novel members of protein families are present across eukaryotes but have been lost in humans. These proteins may well hold clues to understanding differences in cellular function in organisms that are of pressing importance for planetary health.