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Journal Article•DOI•

Proteomic analysis of the acidocalcisome, an organelle conserved from bacteria to human cells.

Guozhong Huang1, Paul N. Ulrich2, Melissa Storey1, Darryl Johnson1, Julie Tischer1, Javier A. Tovar2, Silvia N.J. Moreno1, Ron Orlando1, Roberto Docampo1 •
University of Georgia1, Georgia State University2
11 Dec 2014-PLOS Pathogens (Public Library of Science)-Vol. 10, Iss: 12
TL;DR: A comprehensive insight is provided into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalCisomes are especially adapted for the accumulation of polyphosphate is provided.
Abstract: Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate.

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Citations
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Journal Article•DOI•
Inorganic Polyphosphates As Storage for and Generator of Metabolic Energy in the Extracellular Matrix.

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Werner E.G. Müller1, Heinz C. Schröder1, Xiaohong Wang1•
University of Mainz1
18 Nov 2019-Chemical Reviews
TL;DR: A critical overview about the formation and function of this unique polymer that is capable of storing (bio)chemically useful energy is given.
Abstract: Inorganic polyphosphates (polyP) consist of linear chains of orthophosphate residues, linked by high-energy phosphoanhydride bonds. They are evolutionarily old biopolymers that are present from bacteria to man. No other molecule concentrates as much (bio)chemically usable energy as polyP. However, the function and metabolism of this long-neglected polymer are scarcely known, especially in higher eukaryotes. In recent years, interest in polyP experienced a renaissance, beginning with the discovery of polyP as phosphate source in bone mineralization. Later, two discoveries placed polyP into the focus of regenerative medicine applications. First, polyP shows morphogenetic activity, i.e., induces cell differentiation via gene induction, and, second, acts as an energy storage and donor in the extracellular space. Studies on acidocalcisomes and mitochondria provided first insights into the enzymatic basis of eukaryotic polyP formation. In addition, a concerted action of alkaline phosphatase and adenylate kinase proved crucial for ADP/ATP generation from polyP. PolyP added extracellularly to mammalian cells resulted in a 3-fold increase of ATP. The importance and mechanism of this phosphotransfer reaction for energy-consuming processes in the extracellular matrix are discussed. This review aims to give a critical overview about the formation and function of this unique polymer that is capable of storing (bio)chemically useful energy.

87 citations

Journal Article•DOI•
Polyphosphate: A Multifunctional Metabolite in Cyanobacteria and Algae.

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Emanuel Sanz-Luque1, Emanuel Sanz-Luque2, Devaki Bhaya1, Arthur R. Grossman1•
Carnegie Institution for Science1, University of Córdoba (Spain)2
26 Jun 2020-Frontiers in Plant Science
TL;DR: Polyphosphate (polyP), a polymer of orthophosphate of varying lengths, has been identified in all kingdoms of life It can serve as a source of chemical bond energy (phosphoanhydride bond) as discussed by the authors.
Abstract: Polyphosphate (polyP), a polymer of orthophosphate (PO4 3-) of varying lengths, has been identified in all kingdoms of life It can serve as a source of chemical bond energy (phosphoanhydride bond) that may have been used by biological systems prior to the evolution of ATP Intracellular polyP is mainly stored as granules in specific vacuoles called acidocalcisomes, and its synthesis and accumulation appear to impact a myriad of cellular functions It serves as a reservoir for inorganic PO4 3- and an energy source for fueling cellular metabolism, participates in maintaining adenylate and metal cation homeostasis, functions as a scaffold for sequestering cations, exhibits chaperone function, covalently binds to proteins to modify their activity, and enables normal acclimation of cells to stress conditions PolyP also appears to have a role in symbiotic and parasitic associations, and in higher eukaryotes, low polyP levels seem to impact cancerous proliferation, apoptosis, procoagulant and proinflammatory responses and cause defects in TOR signaling In this review, we discuss the metabolism, storage, and function of polyP in photosynthetic microbes, which mostly includes research on green algae and cyanobacteria We focus on factors that impact polyP synthesis, specific enzymes required for its synthesis and degradation, sequestration of polyP in acidocalcisomes, its role in cellular energetics, acclimation processes, and metal homeostasis, and then transition to its potential applications for bioremediation and medical purposes

78 citations

Journal Article•DOI•
CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor.

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Noelia Lander1, Miguel A. Chiurillo1, Melissa Storey2, Anibal E. Vercesi1, Roberto Docampo1, Roberto Docampo2 •
State University of Campinas1, University of Georgia2
02 Dec 2016-Journal of Biological Chemistry
TL;DR: Endogenously tagged TcIP3R showed co-localization with antibodies against VP1 to acidocalcisomes of Trypanosoma brucei, suggesting that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.

65 citations

Cites background from "Proteomic analysis of the acidocalc..."

  • ...endogenous tagged protein (13) and specific antibodies against the protein (14), as well as proteomic and functional analyses (13, 14)....

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  • ...brucei was demonstrated by endogenous gene tagging (13), by studies using specific antibodies (14), and by proteomic (14) and functional (13) studies....

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Journal Article•DOI•
Acidocalcisomes of eukaryotes.

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Roberto Docampo1, Guozhong Huang1•
University of Georgia1
01 Aug 2016-Current Opinion in Cell Biology
TL;DR: Acidocalcisomes of several unicellular eukaryotes possess a variety of transporters, channels and pumps implying a large energetic requirement for their maintenance and suggesting other important functions waiting to be discovered.

64 citations

Cites background from "Proteomic analysis of the acidocalc..."

  • ...The presence of a V-HATPase, a V-H-PPase, Vtc1, and Vtc4, and the IP3R was also confirmed in these studies, as well as of an enzyme involved in pyrophosphate and polyphosphate metabolism, the vacuolar soluble pyrophosphatase (VSP), and an acid phosphatase [41]....

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  • ...Proteomic analyses of acidocalcisomes of trypanosomatids [40,41] identified several cation transporters, such as putative Zn transporters, an orthologue to the vacuolar iron transporter (VIT) of plants and the Ca-sensitive cross-complementer 1 (CCC1) of the yeast vacuole, which are involved in iron and manganese sequestration, an orthologue to the phosphate-sodium symporter Pho91 of the yeast vacuole, involved in Na and phosphate release, and an orthologue to a polyamine transporter, suggesting roles in inorganic and organic cation uptake, and phosphorus release [41]....

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Journal Article•DOI•
Calcium signaling in trypanosomatid parasites.

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Roberto Docampo1, Roberto Docampo2, Guozhong Huang2•
State University of Campinas1, University of Georgia2
01 Mar 2015-Cell Calcium
TL;DR: The mitochondrial Ca(2+) transporters of trypanosomatids have an important role in the regulation of cell bioenergetics and flagellar Ca( 2+) appears to have roles in sensing the environment.

61 citations

Cites background or methods from "Proteomic analysis of the acidocalc..."

  • ...The acidocalcisome localization of the TbIP3R supports a role for these organelles in Ca2+ signaling....

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  • ...The functional study of TbIP3R [4, 5] and TcIP3R [54] was done by stable transfection of the respective genes in a chicken B lymphocyte cell line devoid of the genes for all three vertebrate IP3Rs (DT40-3KO)....

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  • ...TbIP3R and TcIP3R1 localize to the ER of DT40KO cells, and IP3 is able to release Ca 2+ from these permeabilized cells, from their microsomal vesicles, or from intact cells stimulated by anti-B cell receptor monoclonal antibodies [4, 54, 5]....

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  • ...Knockdown of TbIP3R expression by induction of RNAi results in growth defects in both bloodstream and procyclic trypomastigotes [4, 5]....

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  • ...The acidocalcisome localization was first reported using the epitope-tagged protein [4, 5] and recently confirmed using specific antibodies against TbIP3R [67]....

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References
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Journal Article•DOI•
Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes

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Anders Krogh1, B. Larsson1, G. von Heijne2, Erik L. L. Sonnhammer3•
Technical University of Denmark1, Stockholm University2, Karolinska Institutet3
19 Jan 2001-Journal of Molecular Biology
TL;DR: A new membrane protein topology prediction method, TMHMM, based on a hidden Markov model is described and validated, and it is discovered that proteins with N(in)-C(in) topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for N(out)-C-in topologies.

11,453 citations

Journal Article•DOI•
Improved Prediction of Signal Peptides: SignalP 3.0

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Jannick Dyrløv Bendtsen1, Henrik Nielsen1, Gunnar von Heijne2, Søren Brunak1•
Technical University of Denmark1, Stockholm University2
16 Jul 2004-Journal of Molecular Biology
TL;DR: Improvements of the currently most popular method for prediction of classically secreted proteins, SignalP, which consists of two different predictors based on neural network and hidden Markov model algorithms, where both components have been updated.

6,492 citations

"Proteomic analysis of the acidocalc..." refers methods in this paper

  • ...The membrane topology and presence of signal peptides and was predicted using the following tools: SignalP3 [95], TMHMM2....

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Journal Article•DOI•
A statistical model for identifying proteins by tandem mass spectrometry.

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Alexey I. Nesvizhskii1, Andrew Keller1, Eugene Kolker1, Ruedi Aebersold1•
Institute for Systems Biology1
15 Jul 2003-Analytical Chemistry
TL;DR: A statistical model is presented for computing probabilities that proteins are present in a sample on the basis of peptides assigned to tandem mass (MS/MS) spectra acquired from a proteolytic digest of the sample, and it is shown to produce probabilities that are accurate and have high power to discriminate correct from incorrect protein identifications.
Abstract: A statistical model is presented for computing probabilities that proteins are present in a sample on the basis of peptides assigned to tandem mass (MS/MS) spectra acquired from a proteolytic digest of the sample. Peptides that correspond to more than a single protein in the sequence database are apportioned among all corresponding proteins, and a minimal protein list sufficient to account for the observed peptide assignments is derived using the expectation−maximization algorithm. Using peptide assignments to spectra generated from a sample of 18 purified proteins, as well as complex H. influenzae and Halobacterium samples, the model is shown to produce probabilities that are accurate and have high power to discriminate correct from incorrect protein identifications. This method allows filtering of large-scale proteomics data sets with predictable sensitivity and false positive identification error rates. Fast, consistent, and transparent, it provides a standard for publishing large-scale protein identif...

4,544 citations

"Proteomic analysis of the acidocalc..." refers methods in this paper

  • ...When variants of similar proteins are indistinguishable from peptide data, the ProteinProphet [30] algorithm utilized by the ProteoIQ software treats these identifications as a single protein (a protein ‘‘group’’)....

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  • ...The ProteinProphet algorithm utilized by ProteoIQ software combines hit proteins with degenerate peptide fingerprints into a single identification (a protein ‘‘group’’) and generates a group probability....

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Journal Article•DOI•
Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.

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Olof Emanuelsson1, Henrik Nielsen2, Søren Brunak2, Gunnar von Heijne1•
Stockholm University1, Technical University of Denmark2
21 Jul 2000-Journal of Molecular Biology
TL;DR: A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed and it is estimated that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%.

4,268 citations

Additional excerpts

  • ...1 [91], pTARGET [92], SLPLOCAL [93], and WoLF-PSORT [94]....

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Journal Article•DOI•
WoLF PSORT: protein localization predictor.

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Paul Horton1, Keun Joon Park2, Takeshi Obayashi2, Naoya Fujita2, Hajime Harada2, C. J. Adams-Collier2, Kenta Nakai2 •
National Institute of Advanced Industrial Science and Technology1, Centers for Disease Control and Prevention2
01 Jul 2007-Nucleic Acids Research
TL;DR: WoLF PSORT converts protein amino acid sequences into numerical localization features; based on sorting signals, amino acid composition and functional motifs such as DNA-binding motifs, which allows a user to understand the evidence (or lack thereof) behind the predictions made for particular proteins.
Abstract: WoLF PSORT is an extension of the PSORT II program for protein subcellular location prediction. WoLF PSORT converts protein amino acid sequences into numerical localization features; based on sorting signals, amino acid composition and functional motifs such as DNA-binding motifs. After conversion, a simple k-nearest neighbor classifier is used for prediction. Using html, the evidence for each prediction is shown in two ways: (i) a list of proteins of known localization with the most similar localization features to the query, and (ii) tables with detailed information about individual localization features. For convenience, sequence alignments of the query to similar proteins and links to UniProt and Gene Ontology are provided. Taken together, this information allows a user to understand the evidence (or lack thereof) behind the predictions made for particular proteins. WoLF PSORT is available at wolfpsort.org

2,878 citations

Additional excerpts

  • ...To identify likely contaminants from non-acidocalcisomal compartments in our proteomic dataset, we used a series of subcellular prediction algorithms: TargetP 1.1 [91], pTARGET [92], SLPLOCAL [93], and WoLF-PSORT [94]....

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  • ...1 [91], pTARGET [92], SLPLOCAL [93], and WoLF-PSORT [94]....

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Related Papers (5)
Acidocalcisomes ? conserved from bacteria to man

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01 Mar 2005-Nature Reviews Microbiology
Roberto Docampo, Roberto Docampo, Wanderley de Souza, Kildare Miranda, Kildare Miranda, Peter Rohloff, Silvia N.J. Moreno, Silvia N.J. Moreno 
Rapid Changes in Polyphosphate Content within Acidocalcisomes in Response to Cell Growth, Differentiation, and Environmental Stress inTrypanosoma cruzi

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13 Jul 2001-Journal of Biological Chemistry
Felix A. Ruiz, Claudia O. Rodrigues, Roberto Docampo
Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei

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15 Nov 1994-Biochemical Journal
Anibal E. Vercesi, Silvia N.J. Moreno, Roberto Docampo
Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase.

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24 Apr 2009-Science
Michael Hothorn, Heinz Neumann, Esther D. Lenherr, Mark Wehner, Vladimir Rybin, Paul O. Hassa, Andreas Uttenweiler, Monique Reinhardt, Andrea Schmidt, Jeanette Seiler, Andreas G. Ladurner, Christian Herrmann, Klaus Scheffzek, Andreas Mayer, Andreas Mayer 
Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi.

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15 Sep 1995-Biochemical Journal
Roberto Docampo, David A. Scott, Anibal E. Vercesi, Silvia N.J. Moreno