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WO2005003160A1 - Mutants d'actine - Google Patents

Mutants d'actine Download PDF

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Publication number
WO2005003160A1
WO2005003160A1 PCT/EP2004/051310 EP2004051310W WO2005003160A1 WO 2005003160 A1 WO2005003160 A1 WO 2005003160A1 EP 2004051310 W EP2004051310 W EP 2004051310W WO 2005003160 A1 WO2005003160 A1 WO 2005003160A1
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Prior art keywords
actin
mutants
binding
mutant
residues
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Inventor
Christophe Ampe
Davy Waterschoot
Heidi Rommelaere
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Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin

Definitions

  • the present invention relates to actin mutants, preferably conformational stable actin mutants, comprising at least three, preferably at least five consecutive alanine residues.
  • the invention relates further to the use of these mutants to study the interaction with proteins of the actin folding machinery and with actin binding proteins in general and to study copolymerisation.
  • Actin is the major component of the micr ⁇ filament system of eukaryotic organisms, as such, it plays a central role in cell motility processes. Actin is one of the most conserved proteins known today, and it is thought that its conserved nature reflects its strict structural requirement to interact with itself as well as with a variety of other proteins that modulate the transition between and the dynamics of the monomeric and polymeric state.
  • actin binding proteins such as DNAse I, profilin I, gelsolin and the vitamin D-binding protein
  • co-crystal structures exist (Kabsch et al., 1990; Schutt et al., 1993; McLaughlin et al., 1993; Robinson et al., 1999 ; Otterbein et al., 2002 ).
  • actin modulator proteins like thymosin ⁇
  • the contact sites within actin have been proposed based on cross-linking and /or electron microscopy (EM) data (Safer et al., 1997; Ballweber et al., 2002).
  • EM electron microscopy
  • a first aspect of the invention is a human actin mutant, comprising at least three consecutive alanine residues.
  • said alanine residues are non- endogenous.
  • said actin mutant is a mutant of human beta-actin, with a wild type sequence as shown in SEQ ID N°1. Even more preferably, said actin mutant occurs in a stable conformation.
  • Another aspect of the invention is an actin mutant comprising at least five consecutive alanine residues.
  • said alanine residues are non- endogenous.
  • said actin mutant is a mutant of mammalian beta-actin, even more preferably said mutant is a mutant of human beta-actin with a wild type sequence as shown in SEQ ID N°1. Even more preferably, said actin mutant occurs in a stable conformation.
  • Still another aspect of the invention is the use of an actin mutant according to the invention to study the interaction of actin with actin binding proteins.
  • An actin binding protein may be any protein that interacts with actin.
  • said actin binding protein may be a protein of the acting folding machinery.
  • Still another aspect of the invention is the use of an actin mutant according to the invention to study copolymerisation.
  • Fig. 1 Actin alanine mutants, folding capacity and binding to actin-binding proteins.
  • A Autoradiogram of a native gel analysis of ⁇ S-labeled ⁇ -actin or ⁇ -actin 60(A5) produced in an in vitro transcription translation reaction (control), band shifted with either thymosin ⁇ 4, DNAse I or adseverin. Band-shifts are also generated for interactions with relatively low Kd-values (e.g. thymosin ⁇ 4), it is however important that the actin-binding protein is, like actin, attracted by the anode.
  • B 3D-structure of the actin fold (Kabsch et al. 1990) with in green those regions that, when mutated, cause folding defective mutants. In the right panel the actin molecule is rotated by 180°. ATP is in yellow.
  • CAP binds in the subdomain 1-3 cleft of actin and mutating residues involved in ATP-binding stabilizes the CAP-actin interaction
  • A Autoradiogram of a native gel analysis without ATP of 35 S-labeled actin (lane 1) and actin 155(A5) (lanes 2-4) produced in an in vitro transcription translation reaction.
  • DNAse I can form a ternary complex with the actin-CAP complex (lane 3, 0.25 nM DNAsel added) or can compete with CAP for actin binding when added at higher concentrations (lane 4, 8.25 nM DNAse I added).
  • CAP is dimeric, hence can bind two actin molecules resulting in two shifts with DNAse I.
  • FIG.4 Actin residues important for polymerisation.
  • A-B 3D-representation of actin with highlighted in pink the residues important in the F- actin contacts according to the two F-actin models.
  • A in the right-handed helix model of Holmes et al. (1990), interactions between molecules along one strand involve contacts between subdomain 4 and subdomain 2 of one protomer with subdomain 3 of the next, i.e. residues 243-245 contact 322-325, 202-204 contact 286-289 and 41-45 contact 166-169 and 375 (blue circles).
  • Arg 39 is possibly involved in a salt bridge with Asp 286 and GIu 270, each in a different actin molecule.
  • FIG. 5 In vivo evidence for the Holmes Model. Expression of N-terminally myc-tagged actin alanine mutants in NIH 3T3 cells. Western blotting of lysates of transfected cells and probing with mouse anti-myc antibodies revealed the presence of actins with the correct molecular weight.
  • A Wild type actin
  • B actin 345(A5)
  • C actin 60(A5)
  • D actin 35
  • E actin 285(A5)
  • F actin 145(A5)
  • G actin 295(A5)
  • H actin 95(A5)
  • I actin 350(A5).
  • FIG. 6 Introduction of non-copolymerising actin mutants in cells has drastic effects on the cytoskeleton and cell shape.
  • A-C Details of three different transfected NIH3T3 cells with myc-tagged actin mutants 60(A5), (A) is a detail of Figure 5C. Green is anti-myc, red is phallo ⁇ din and blue is DAPI staining (only in C). Actin filaments (red) appear as extra-nuclear short thick fibers, the nucleus seems to be smaller and deformed.
  • Bind(ing) means any interaction, be it direct or indirect.
  • a direct interaction implies a contact between the binding partners.
  • An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two compounds. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more compounds
  • Endogenous residues are residues that occur as such in the wild type protein.
  • Non- endogenous residues are residues that are mutated when compared with the residue ate the same position in the wild type protein.
  • a mutant that occurs in stable conformation as used here means that at least a part of the population is folded, as judged on non-denaturing protein gel. Criteria for the stable conformation are that (1) the mutant gives a band in the non-denaturing gel that runs in a similar way as wilt type actin and (2) the mutant shows a band shift with at least on actin binding protein.
  • N-terminal myc-tagged wild type and actin mutants were made by PCR using the actin-alanine mutants in the pcDNA3.1 vector as template, a 5' primer containing the myc- sequence preceded by a Hindi 11 site and a 3' primer containing a Xbal site. These fragments were ligated in Hindlll-Xbal digested pcDNA3.1. Constructs were sequenced at the 5' and /or 3' end of their coding sequence and the alanine-scan mutants at the site of the introduced mutations.
  • actin alanine mutants were expressed as 35 S-labeled proteins in in vitro transcription translation reactions in reticulocyte lysate (Promega) according to the manufacturer's instructions, using 0.02 ⁇ Ci ⁇ S-methionine (ICN) and 200 ng DNA per 25 ⁇ l reaction. After incubation at 30°C for 1.5 hour, we analysed the reaction products on denaturing tricine gels (Schagger and von Jagow, 1987) and on non -denaturing polyacrylamide gels according to Safer (1989) with ATP, followed by auto radiography. To monitor CAP-binding, ATP was omitted from the gels.
  • the amount of ⁇ S-actin bound to CAP was quantified by phosphor imaging (Typhoon 9200 variable mode imager, Amersham Biosciences) and the ImageQuant software package.
  • 1 ⁇ l of the respective actin binding proteins was added to 3 ⁇ l of the in vitro transcription translation reaction. After 1 minute incubation the mixture was analysed on native gels with 200 ⁇ M ATP, or in the case of adseverin with 200 ⁇ M ATP and 200 ⁇ M Ca 2+ .
  • the final concentration of the respective actin binding proteins was 2 ⁇ M for DNAse I, 12.5 ⁇ M for thymosin ⁇ 4 and 1 ⁇ M for adseverin.
  • concentrations are the minimal amounts of the actin binding proteins needed to cause a band shift of wild type actin, as was determined by a concentration series. Binding capacity was inspected by eye. Reduced or less effective binding means that only a part of the population of the mutant actin molecules shifted, and part remain unshifted.
  • DNAse I was purchased from Worthington. Thymosin ⁇ 4 was chemically synthesized on a model 431A peptide synthesizer using solid phase Fmoc chemistry. Adseverin was recombinantly expressed and purified according to Robbens et al. (1998).
  • Example 1 The alanine-scan mutants allow probing interfaces with actin binding proteins
  • Example 2 An extended interface with thymosin ⁇ 4 Next we embarked on investigating proteins of which the interface with actin is controversial or unknown. So far, it has been impossible to obtain actin thymosin ⁇ crystals, but various models exist for the actin thymosin ⁇ interaction.
  • the band-shift assay results show compromised binding for actins mutated in the regions 35-39, 60-74, 140-149, 194-198, 204- 209, 300-304, 335-339 and 350-354 (Figure 2C).
  • Example 3 Cyclase associated protein, CAP, interacts with actin in the cleft between subdomains 1 and 3.
  • the band shift assays above were performed in the presence of ATP.
  • ATP is omitted from the native gels, a new 35 S-labeled actin species, representing a complex between actin and endogenous CAP, appears in between the CCT-actin complex and hemoglobin ( Figure 3A, lane 1; McCormack et al., 2001 ; mass-spectrometry showed this is indeed an actin-CAP complex, data not shown).
  • Figure 3A lane 1; McCormack et al., 2001 ; mass-spectrometry showed this is indeed an actin-CAP complex, data not shown.
  • Example 4 Actins mutated in the ATP binding pocket remain CAP-arrested
  • CAP binding is increased for actin mutants 70(A5) 155(A5), 160(A5), 179(A5), 210(A5) and 300(A5) ( Figure 3C, Table I).
  • the latter five even bind to CAP when ATP is present in the gels, whereas under these conditions, no complex is observed for wild type actin and other folding capable mutants.
  • CAP binding is sensitive to the nucleotide state of actin, hence CAP may be an actin binding protein involved in the stabilization of nucleotide free actin.
  • CAP may be an actin binding protein involved in the stabilization of nucleotide free actin.
  • a more active contribution of CAP in the actin folding pathway could be that it acts as an ATP-loading machine post-CCT.
  • the actin alanine mutants provide important information on the actin interaction site with CAP and suggest clues on CAP function.
  • Example 5 In vitro actin polymerisation supports the Holmes F-actin model Since we could locate regions in actin important for interaction with actin binding proteins, we hypothesized that we could also identify actin-actin contacts between neighboring protomers in the actin filament.
  • Two major F-actin models have been proposed which we will refer to as the Holmes model and the ribbon model.
  • the first model is based on low-resolution diffraction data of oriented F-actin filaments (Holmes et al., 1990) and is widely accepted because it is consistent with numerous biochemical experiments (e.g. Steinmetz et al., 1998).
  • the ribbon model is intriguing because it is observed in the high-resolution profilin- actin crystal structure (Schutt et al., 1993).
  • actin protomers are associated in two strings wound around each other in a right-handed helix, with interactions along and across the strands.
  • the interactions between molecules along one strand involve contacts between subdomain 4 and subdomain 2 of one protomer with subdomain 3 of the next.
  • Contacts across the two strands are made between subdomain 1 of one protomer and subdomain 4 of the other and by the so-called hydrophobic plug which inserts into a hydrophobic pocket (Holmes et al., 1990; for a detailed list of residues involved see legend of Figure 4A).
  • mutants in the region 45-49, 50-54, 95-99, 130-134 and 350-354, characteristic for the contacts in the ribbon model display wild type or nearly wild type copolymerisation capacity (mutants 85(A5), 90(A5) and 355(A5) do not fold).
  • mutants 85(A5), 90(A5) and 355(A5) do not fold.
  • actin mutants 35(A5) en 285(A5) appear to act as filament capping molecules when transfected in eukaryotic cells (see below), suggesting actin 35(A5) is crucial at the pointed and actin 285(A5) at the barbed end, which can only be explained in the Holmes model.
  • mutants 275(A5), 280(A5), 290(A5) and 295(A5) are close to the 285-289 loop and mutants 145(A5) and 150(A5) comprise a core ⁇ -strand in subdomain 3 that is close to the predicted Holmes contact regions 166-169 and 171-173.
  • mutants 145(A5) and 150(A5) comprise a core ⁇ -strand in subdomain 3 that is close to the predicted Holmes contact regions 166-169 and 171-173.
  • mutating the region 220-224 may influence correct positioning of the hydrophobic plug.
  • the reduced copolymerisation capacity of 184(A5), having mutations in the core of a helix can be explained this way.
  • the copolymerisation assay above inherently uses a large excess of carrier wild type actin and does not allow investigation of more subtle effects on actin filaments. Moreover, as yet, there is no evidence that in vivo polymer contacts are the same as those predicted from the models (Mounier and Sparrow, 1997). Therefore we investigated the behavior of selected myc-tagged actin mutants in NIH3T3, NG108 or Hek293T cells. Only the results for NIH3T3 cells are shown, but all cell types gave similar results. The actin cytoskeleton was stained with phallo ⁇ din-alexa red, and the actin mutants were detected with an anti-myc FITC antibody.
  • Mutants 60(A5) and 35(A5) induce in many cases formation of densely stained actin fibers in the center of the cells with some residual F-actin staining in a punctuate pattern in case of mutant 35(A5) ( Figure 5C, 5D and 6).
  • the dense short actin fibers formed in 60(A5) transfected cells are not in. the nucleus, as can be seen in Figure 6C.
  • an interaction with an organizing actin binding protein may be compromised, but perhaps more likely, the disturbance of the cytoskeleton is caused by a continuously filament capping action of these mutants.
  • regions 35-49 and 60-64, and 285 ⁇ 289 are on opposite sides of the actin protomer and, if located in an F-actin contact, mutants in these regions may behave as actin filament capping proteins and will affect actin dynamics with pointed end mutants having a stronger effect.
  • the Holmes model predicts that regions 35-39 and 60-64 are at the minus end and 285-289 is at the plus end, our in vivo data again support this model.
  • Tables Table I Summary of the folding capacity of actin alanine mutants and binding to DNAse I, adseverin, thymosin ⁇ 4 and CAP. * + indicates that the mutant folds or interacts with the listed binding protein, (+) indicates only a small part of the population is folded, - and highlighted in light gray indicates lack of folding or binding, +/- is reduced binding, ++ and +++ highlighted in dark gray indicates increased CAP binding.
  • mutant aa changed folds* DH&se I* Adsev* Thym b4* CAP* actb (WT) actb2 (A3) actb5(A5) IAALV actb!0(A5) VDNGS actbl5(A5) GMCKA weak actb20( ⁇ 5) GFAGD actb25(A5) DAP A weak actb30(A5) VFPSI weak actb35(A5) VGRPR +/- actb40(A5) HQGVM weak actb45 (A5) VGMGQ +/- actb50(A5) KDSYV weak actb55(A5) GDEAQ actb60(A5) SKRGI actb65(A5) Ii L Y +/- actb70(A5) PIEHG actb75(A5) IVTNW actb80(A5) DDMCK actb85(A5) IWHHT actb90(A5) FYNE weak actb95
  • actb220(A5) ⁇ actb225(AS) actb230(A5) actb235(A5) actb240(A5) actb245(A5) actb250(A5) actb265(A5)
  • Thymosin-beta(4) changes the conformation and dynamics of actin monomers. Biophys.J. 78, 2516-2527.
  • Eukaryotic cytosolic chaperonin contains t-complex polypeptide 1 and seven related subunits. Proc.Natl.Acad.Sci.U.S.A 90, 11975-11979.
  • Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends. Biochemistry 36, 5806-5816.

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Abstract

La présente invention concerne des mutants d'actine, de préférence des mutants d'actine stables de conformation, qui comprenne au moins trois, et de préférence au moins cinq résidus alanine consécutifs. Cette invention concerne aussi l'utilisation de ces mutants pour étudier l'interaction avec des protéines de la machinerie de repliement de l'actine avec des protéines associées à l'actine en général et pour étudier la copolymérisation. .
PCT/EP2004/051310 2003-07-01 2004-06-30 Mutants d'actine Ceased WO2005003160A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140037577A1 (en) * 2006-10-27 2014-02-06 Michael A. Zeligs Anti-parasitic methods and compositions utilizing diindolylmethane-related indoles
WO2024262350A1 (fr) * 2023-06-19 2024-12-26 サントリーホールディングス株式会社 Peptide ou sel de celui-ci, composition le contenant, et composition pour supprimer le déclin ou améliorer les fonctions cognitives

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AYSCOUGH KATHRYN R ET AL: "ACTIN: General principles from studies in yeast", ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY ANNUAL REVIEWS INC. {A}, P.O. BOX 10139, 4139 EL CAMINO WAY, PALO ALTO, CALIFORNIA 94306, USA SERIES : ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY (ISSN 1081-0706), 1996, pages 129 - 160, XP002307840, ISSN: 0-8243-3112-5 *
MCCORMACK E A ET AL: "Mutational screen identifies critical amino acid residues of beta-actin mediating interaction between its folding intermediates and eukaryotic cytosolic chaperonin CCT.", JOURNAL OF STRUCTURAL BIOLOGY. AUG 2001, vol. 135, no. 2, August 2001 (2001-08-01), pages 185 - 197, XP002307838, ISSN: 1047-8477 *
ROMMELAERE HEIDI ET AL: "Structural plasticity of functional actin: pictures of actin binding protein and polymer interfaces.", STRUCTURE (CAMBRIDGE, MASS. : 2001) OCT 2003, vol. 11, no. 10, October 2003 (2003-10-01), pages 1279 - 1289, XP002307837, ISSN: 0969-2126 *
WERTMAN K F ET AL: "Systematic mutational analysis of the yeast ACT1 gene.", GENETICS. OCT 1992, vol. 132, no. 2, October 1992 (1992-10-01), pages 337 - 350, XP002307839, ISSN: 0016-6731 *
XIA D ET AL: "Deletion of amino acids from the carboxy-terminal end of actin.", CELL MOTILITY AND THE CYTOSKELETON. 1995, vol. 32, no. 3, 1995, pages 163 - 172, XP008039579, ISSN: 0886-1544 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140037577A1 (en) * 2006-10-27 2014-02-06 Michael A. Zeligs Anti-parasitic methods and compositions utilizing diindolylmethane-related indoles
WO2024262350A1 (fr) * 2023-06-19 2024-12-26 サントリーホールディングス株式会社 Peptide ou sel de celui-ci, composition le contenant, et composition pour supprimer le déclin ou améliorer les fonctions cognitives

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