1. |
- Tavella, T A, et al.
(author)
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Yeast-based high-throughput screens for discovery of kinase inhibitors for neglected diseases.
- 2021
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In: Advances in Protein Chemistry and Structural Biology. - : Elsevier. - 1876-1631. ; 124, s. 275-309
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Research review (peer-reviewed)abstract
- The discovery and development of a new drug is a complex, time consuming and costly process that typically takes over 10 years and costs around 1 billion dollars from bench to market. This scenario makes the discovery of novel drugs targeting neglected tropical diseases (NTDs), which afflict in particular people in low-income countries, prohibitive. Despite the intensive use of High-Throughput Screening (HTS) in the past decades, the speed with which new drugs come to the market has remained constant, generating doubts about the efficacy of this approach. Here we review a few of the yeast-based high-throughput approaches that can work synergistically with parasite-based, in vitro, or in silico methods to identify and optimize novel antiparasitic compounds. These yeast-based methods range from HTP screens to identify novel hits against promising parasite kinase targets to the identification of potential antiparasitic kinase inhibitors extracted from databases of yeast chemical genetic screens.
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2. |
- Seely, Savannah M., et al.
(author)
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Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome
- 2023
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Journal article (other academic/artistic)abstract
- Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3’-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.
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3. |
- Allison, Timothy M., et al.
(author)
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Complementing machine learning‐based structure predictions with native mass spectrometry
- 2022
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In: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:6
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Journal article (peer-reviewed)abstract
- The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.
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4. |
- Panagaki, Dimitra, et al.
(author)
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Nuclear envelope budding is a response to cellular stress.
- 2021
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 118:30
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Journal article (peer-reviewed)abstract
- Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.
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5. |
- Sahin, Cagla, et al.
(author)
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Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization
- 2022
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:27, s. 11949-11954
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Journal article (peer-reviewed)abstract
- α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinsonâs disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of â2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
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6. |
- Sridhara, Sagar, 1989
(author)
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Multiple structural flavors of RNase P in precursor tRNA processing.
- 2024
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In: Wiley interdisciplinary reviews. RNA. - 1757-7012. ; 15:2
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Journal article (peer-reviewed)abstract
- The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
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7. |
- Wu, Fei, 1993-
(author)
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Exploring Membrane Proteins within the Inner Mitochondrial and Endoplasmic Reticulum Membranes : Mitochondrial respiratory complexes and ER-localized Shr3
- 2024
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Doctoral thesis (other academic/artistic)abstract
- Membrane proteins play important roles in various life processes, for example, those in the inner mitochondrial membrane (IMM), endoplasmic reticulum (ER) membrane, and plasma membrane (PM). Oxidative phosphorylation complexes, densely packed in the IMM are crucial for energy transduction in eukaryotes. We determined three entire II2III2 IV2 supercomplex (SC) structures with 114 lipids at 2.1-2.4 Å resolution in Perkinsus marinus (P. marinus). The structures show a complete electron transfer pathway from complex II (CII) to complex IV (CIV). These architectures also reveal rotation states of the iron sulfur protein (ISP) in complex III (CIII), from one of which we observed two novel proteins that might impair the electron transfer. We also studied how the salt concentration and detergent affect the electron transfer. We determined the SC III2 IV-cytochrome c (cyt. c) cryo-EM structure at 20 mM salt concentration condition. Together with kinetic study, these data implicate that multiple cyt. c are involved in electron transfer between CIII and CIV. Our kinetic studies of CIV also indicate a native ligand bound near its K proton pathway which could be removed by detergent, leading to an increase in electron transfer rate and the activity of the enzyme. Most biogenesis of integral membrane proteins in eukaryotes is done in ER, such as the amino acid permeases (AAP), which function as amino acid transporters in the PM. Its synthesis and functional folding in Saccharomyces cerevisiae (S. cerevisiae) requires an ER membrane-localized chaperone, Shr3. We utilized a yeast growth-based genetic assay, in conjunction with a split-ubiquitin yeast two-hybrid assay, to demonstrate the selective interaction between Shr3 and nested C-terminal AAP truncations. This interaction exhibited a distinct pattern, wherein it gradually intensified and then weakened as more transmembrane helices folded. The work presented in this thesis contributions to an increased understanding of the organization and function of SCs, the effects of protein subunits, salt concentrations, and detergents on electron transfer, as well as the mechanism of Shr3 on AAP folding in the ER membrane. Together, these works have shed light on the understanding of the structure and function of several membrane proteins.
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8. |
- Sendker, Franziska L., et al.
(author)
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Emergence of fractal geometries in the evolution of a metabolic enzyme
- 2024
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In: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 628:8009, s. 894-900
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Journal article (peer-reviewed)abstract
- Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2,3,4; however, so far, molecular assembly into fractals is restricted to synthetic systems5,6,7,8,9,10,11,12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpiński triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.
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9. |
- D'Angiolo, M., et al.
(author)
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A yeast living ancestor reveals the origin of genomic introgressions
- 2020
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 587, s. 420-425
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Journal article (peer-reviewed)abstract
- A yeast clonal descendant of an ancient hybridization event is identified and sheds light on the early evolution of the Saccharomyces cerevisiae Alpechin lineage and its abundant Saccharomyces paradoxus introgressions. Genome introgressions drive evolution across the animal(1), plant(2) and fungal(3) kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast(4), has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage(5), which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.
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10. |
- Zeng, Jiao, et al.
(author)
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High-resolution structure of a fish aquaporin reveals a novel extracellular fold
- 2022
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In: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
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Journal article (peer-reviewed)abstract
- Aquaporins are protein channels embedded in the lipid bilayer in cells from all organisms on earth that are crucial for water homeostasis. In fish, aquaporins are believed to be important for osmoregulation; however, the molecular mechanism behind this is poorly understood. Here, we present the first structural and functional characterization of a fish aquaporin; cpAQP1aa from the fresh water fish climbing perch (Anabas testudineus), a species that is of high osmoregulatory interest because of its ability to spend time in seawater and on land. These studies show that cpAQP1aa is a water-specific aquaporin with a unique fold on the extracellular side that results in a constriction region. Functional analysis combined with molecular dynamic simulations suggests that phosphorylation at two sites causes structural perturbations in this region that may have implications for channel gating from the extracellular side.
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