WO2007139846A2 - Expression bactérienne de la hsp20 phosphorylée et leurs réactifs - Google Patents

Expression bactérienne de la hsp20 phosphorylée et leurs réactifs Download PDF

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WO2007139846A2
WO2007139846A2 PCT/US2007/012320 US2007012320W WO2007139846A2 WO 2007139846 A2 WO2007139846 A2 WO 2007139846A2 US 2007012320 W US2007012320 W US 2007012320W WO 2007139846 A2 WO2007139846 A2 WO 2007139846A2
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hsp20
host cell
protein kinase
active
recombinant host
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WO2007139846A3 (fr
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Charles Robert Flynn
Christopher Smoke
Elizabeth Furnish
Padmini Komalavilas
Jeffery Thresher
Colleen Brophy
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Arizona State University ASU
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Arizona State University ASU
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Definitions

  • Smooth muscle relaxation can be induced by a variety of endogenous and pharmacologic agents such as nitric oxide (NO) through cyclic nucleotide signaling cascades. NO released from stimulated endothelial cells or provided by NO donors such as sodium nitroprusside (SNP) diffuses across cell membranes activating guanylyl cyclase, increasing intracellular cGMP concentrations, and activating cGMP-dependent protein kinase [I].
  • SNP sodium nitroprusside
  • An alternate pathway for smooth muscle relaxation involves activation of adenylyl cyclase (by agonists such as forskolin), increasing intracellular cAMP concentrations, and activation of cAMP-dependent protein kinase [2].
  • HSP20 One downstream target of both cyclic nucleotide signaling cascades is the heat shock-related protein 20, HSP20.
  • HSP20 is highly and constitutively expressed in skeletal, cardiac, and smooth muscle and increases in the phosphorylation of HSP20 are associated with cyclic nucleotide-dependent relaxation of vascular smooth muscle [3-5].
  • HSP20 requires phosphorylation at a serine or threonine residue (S 16 in HSP20) to render it functionally active [5,6].
  • HSP20 is a specific substrate protein of PKA and PKG in vivo and accumulating evidence suggests HSP20 has several physiological and biochemical roles including controlling muscle tone, regulating cell motility, modulating actin filament dynamics and protection against intimal hyperplasia and ischemia/reperfusion injury [7-14]. Although the molecular mechanisms of action for HSP20 have not been determined fully, it is an actin-associated protein and we have shown that HSP20 peptide treatment leads to loss of actin stress fibers and disruption of focal adhesion complexes [9-10]. Treatment with a phosphopeptide HSP20 analogs leads to a dissociation and activation of cofilin resulting in depolymerization of the actin cytoskeleton. Collectively, these data suggest HSP20 involvement in thin filament regulatory mechanisms
  • PTDs protein transduction domains
  • the present invention provides recombinant host cells, comprising:
  • a recombinant expression vector operably linked to a nucleic acid sequence encoding a catalytic subunit of a protein kinase selected from the group consisting of protein kinase A and protein kinase G;
  • the present invention provides methods for making phosphorylated HSP20 polypeptide, comprising culturing the recombinant host cells of the first aspect of the invention under conditions to promote expression of the catalytic subunit of the protein kinase and the active HSP20 polypeptide, and to promote phosphorylation of the active HSP20 polypeptide by the expressed protein kinase catalytic subunit.
  • the present invention provides recombinant host cells, comprising:
  • a recombinant expression vector operably linked to a nucleic acid sequence encoding a catalytic subunit of a protein kinase selected from the group consisting of protein kinase A and protein kinase G;
  • the present invention provides methods for treating a condition susceptible to treatment with an active HSP20 polypeptide, comprising administering a recombinant host cell of the invention to a subject in need thereof, wherein the active HSP20 polypeptide produced by the host cell treats the condition.
  • the present invention provides recombinant host cells, comprising:
  • a recombinant expression vector operably linked to a nucleic acid sequence encoding a catalytic subunit of a protein kinase selected from the group consisting of protein kinase A and protein kinase G;
  • a recombinant expression vector operably linked to a nucleic acid sequence encoding an active HSP20 polypeptide.
  • the recombinant host cells of this aspect of the invention can be used, for example, to produce active, phosphorylated HSP20 polypeptides, which can be used for a variety of therapeutic uses, as described below, and can also be used for gene therapy.
  • the recombinant host cells can be prokaryotic or eukaryotic, and can be transiently or stably transfected.
  • Exemplary host cells are bacterial cells (such as E. coli), yeast, and human- derived cells.
  • Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
  • Recombinant expression vector includes vectors that operatively link so as to permit expression of a nucleic acid coding region or gene to any promoter capable of effecting expression of the gene product.
  • the promoter sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive).
  • expression vectors for use in transfecting prokaryotic and eukaryotic cells is also well known in the art, and thus can be accomplished via standard techniques.
  • the expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA.
  • the expression vector comprises a plasmid.
  • the invention is intended to include other expression vectors that serve equivalent functions, such as viral vectors.
  • the recombinant expression vector operably linked to the protein kinase catalytic subunit and to the active HSP20 peptide can be the same recombinant expression vector expressing both.
  • a first recombinant expression vector is operably linked to a nucleic acid sequence encoding a catalytic subunit of a protein kinase selected from the group consisting of protein kinase A and protein kinase G, or catalytic subunits thereof; and a second recombinant expression vector operably linked to a nucleic acid sequence encoding an active HSP20 polypeptide
  • the nucleic acid sequences encoding a catalytic subunit of a protein kinase selected from the group consisting of protein kinase A and protein kinase G can encode full length protein kinase A or G, or may encode only the catalytic subunit.
  • the protein kinase G is the human protein kinase G amino acid sequence comprising or consisting of SEQ ID NO: 45, or catalytic subunit thereof.
  • the protein kinase G is encoded by the nucleic acid sequence comprising or consisting of SEQ ID NO: 46 or SEQ ID NO: 49.
  • the protein kinase A is the human protein kinase A amino acid sequence comprising or consisting of SEQ ID NO: 47, or catalytic subunit thereof.
  • the protein kinase G is encoded by the nucleic acid sequence comprising or consisting of SEQ ID NO: 48.
  • the nucleic acid sequence encoding the protein kinase A or protein kinase C can be any known in the art, including but not limited to those encoding the protein kinases as shown in Table 1.
  • a nucleic acid sequence encoding the catalytic subunit of protein kinase G is used.
  • active HSP20 polypeptide refers to full length HSP20, or any polypeptide portion thereof that can be used to promote smooth muscle relaxation after appropriate phosphorylation (see, for example, the methods disclosed herein for demonstrating the ability to promote smooth muscle relaxation).
  • nucleic acid sequence encoding an active HSP20 polypeptide encodes an active HSP20 polypeptide of general formula I:
  • X2 is 0, I 5 2, 3, or 4 amino acids of the sequence WLRR (SEQ ID NO:7);
  • X3 is 0, I 9 2, or 3 amino acids of a sequence of genus Z1-Z2-Z3, wherein Zl is selected from the group consisting of G and D;
  • Z2 is selected from the group consisting of L and K; and Z3 is selected from the group consisting of S, K and T;
  • X4 is absent or comprises a transduction domain; and wherein u is 1, 2, 3, 4, or 5.
  • u is 1, 2, 3, 4, or 5.
  • at least one of Xl and X4 comprises a transduction domain.
  • nucleic acid sequence encoding an active HSP20 polypeptide encodes an active HSP20 polypeptide of general formula II: X1-X2-ASAPLP-X3-X4 (SEQ ID NO: 8) wherein Xl is absent or comprises a cell transduction domain;
  • X2 is 0-14 amino acids of the sequence of heat shock protein 20 between residues 1 and 14 of SEQ ID NOrI;
  • X3 is 0-140 amino acids of heat shock protein 20 between residues 21 and 160 of SEQ ID NO:1;
  • X4 is absent or comprises a cell transduction domain.
  • At least one of Xl and X4 comprises a transduction domain.
  • nucleic acid sequence encoding an active HSP20 polypeptide encodes an active HSP20 polypeptide of formula: Xl-SEQ ID NO:10-X4 (SEQ ID NO: 9), wherein Xl is absent or comprises a cell transduction domain; wherein (SEQ ID NO: 10) is Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro GIy Leu Lys; and wherein X4 is absent or comprises a cell transduction domain.
  • nucleic acid sequence encoding an active HSP20 polypeptide encodes an active HSP20 polypeptide of general formula III: Xl-[X2-X3-RRASAP]u-X4 (SEQ ID NOs: 11-15) wherein Xl is absent or comprises a transduction domain; X2 is absent or is W; X3 is absent or is L X4 is absent or comprises a transduction domain; and wherein u is 1, 2, 3, 4, or 5.
  • At least one of Xl and X4 comprises a transduction domain.
  • transduction domain means one or more amino acid sequence or any other molecule that can carry the active domain across cell membranes. These domains can be linked to other polypeptides to direct movement of the linked polypeptide across cell membranes. Transduction domains are known to those of skill in the art, and include, but are not limited to: (R) 4-9 (SEQ ID NO: 44)
  • GWTLNSAGYLLGLINLKALAALAKKIL SEQ ID NO: 19
  • PLSSIFSRIGDP SEQ ID NO: 20
  • AAVLLPVLLAAP (SEQ ID NO: 22);
  • VTVLALGALAGVGVG (SEQ ID NO: 23);
  • GALFLGWLGAAGSTMGAWSQP SEQ ID NO: 24
  • GWTLNSAGYLLGLINLKALAALAKKIL SEQ ID NO ⁇ S
  • KAFAKLAARLYRKAGC SEQ ID NO: 28
  • KAFAKLAARLYRAAGC SEQ ID NO: 29
  • AAFAKLAARLYRKAGC SEQ ID NO: 30
  • KAFAALAARLYRKAGC SEQ ID NO: 31
  • KAFAKLAAQLYRKAGC (SEQ ID NO: 32);
  • the transduction domain comprises or consists of a polypeptide of general formula IV:
  • J is any hydrophobic amino acid; wherein Bi, B 2 , and B 3 are independently any basic amino acid; and wherein n is between 1 and 10.
  • n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a preferred embodiment, n is 1, 2, or 3.
  • J 1 -J 4 are independently any hydrophobic amino acid selected from the group consisting of Trp, Tyr, Leu, He, Phe, VaI, Met, Cys, Pro, and Ala; and
  • Bi, B 2 , and B 3 are independently arginine, histidine, or lysine.
  • both B 1 and B 2 are arginine or lysine and B 3 is either lysine or arginine but is not the same as Bi and B 2 .
  • Bj and B 2 are arginine and B 3 is lysine.
  • J1-J4 are independently selected from the group consisting of Trp, Leu, He, and Ala.
  • Ji is Trp, 3% is Leu, J 3 is He, or J 4 is Ala, or any combination thereof.
  • the transduction domains comprises or consists of the amino acid sequence (WLRRIKA) n (SEQ ID NO: 40), wherein n is 1-10.
  • WLRRIKA amino acid sequence
  • the polypeptide can comprise or consist of 1, 2, 3, 4, 5,
  • n 1, 2, or 3.
  • polypeptides include:
  • WLRRIKA SEQ ID NO: 41
  • WLRRIKAWLRRIKA SEQ ID NO: 42
  • WLRRIKAWLRRIKA SEQ ID NO: 42
  • the present invention provides methods for making phosphorylated HSP20 polypeptide, comprising culturing the recombinant host cells of the first aspect of the invention under conditions to promote expression of the catalytic subunit of the protein kinase and the active HSP20 polypeptide, and to promote phosphorylation of the active HSP20 polypeptide by the expressed protein kinase catalytic subunit. All of the various embodiments of the first aspect of the invention also apply to this second aspect of the invention.
  • the present invention provides recombinant host cells, comprising: (a) a recombinant expression vector operably linked to a nucleic acid sequence encoding a catalytic subunit of a protein kinase selected from the group consisting of protein kinase A and protein kinase G; and
  • a recombinant expression vector operably linked to a nucleic acid sequence encoding a polypeptide target of protein kinase A or protein kinase G.
  • the various embodiments of recombinant host cells, recombinant expression vectors, and protein kinase A and G from the first aspect of the invention also apply to the third aspect of the invention.
  • the recombinant expression vector operably linked to the protein kinase catalytic subunit in this third aspect does not encode the full length protein, but only the catalytic subunit.
  • a first recombinant expression vector is operably linked to a nucleic acid sequence encoding the catalytic subunit of a protein kinase selected from the group consisting of protein kinase A and protein kinase G; and a second recombinant expression vector operably linked to a nucleic acid sequence encoding a protein kinase A or protein kinase G target.
  • a nucleic acid sequence encoding the catalytic subunit of protein kinase G is used.
  • any nucleic acid encoding a protein kinase A or G target containing the consensus sequence recongiton site REAS (SEQ ID NO: 50) and capable of being phosphorylated can be used in the recombinant host cells of the invention.
  • the recombinant host cell comprises a bacterial host cell.
  • the present invention provides methods for treating a condition susceptible to treatment with an active HSP20 polypeptide, comprising administering a recombinant host cell of the invention to a subject in need thereof, wherein the active HSP20 polypeptide produced by the host cell treats the condition.
  • transduction domains can induce apoptosis and allow for protein exit from the cell. This is a destructive cellular process.
  • a cell therapy application may be possible wherein host mammalial cells containing the TD expression element would serve as a "biodegradable" implantable/injectable protein production vehicle that would yield active TD-HSP20 upon programmed cell death and lysis.
  • HSP20 polypeptides as therapeutic agents for the following: (a) inhibiting smooth muscle cell proliferation and/or migration; (b) promoting smooth muscle relaxation; (c) increasing the contractile rate in heart muscle; (d) increasing the rate of heart muscle relaxation; (e) promoting wound healing; (f) reducing scar formation; (g) disrupting focal adhesions; (h) regulating actin polymerization; and (i) treating or inhibiting one or more of intimal hyperplasia, stenosis, restenosis, atherosclerosis, smooth muscle cell tumors, smooth muscle spasm, angina, Prinzmetal's angina (coronary vasospasm), ischemia, stroke, bradycardia, hypertension, pulmonary (lung) hypertension, asthma (bronchospasm), toxemia of pregnancy, pre-term labor, pre-eclampsia/eclampsia, Raynaud's disease or phenomenon, hemolytic-uremia, non- occ
  • the invention provides cell therapy methods for one or more of the following therapeutic uses: (a) inhibiting smooth muscle cell proliferation and/or migration; (b) promoting smooth muscle relaxation; (c) increasing the contractile rate in heart muscle; (d) increasing the rate of heart muscle relaxation; (e) promoting wound healing; (f) reducing scar formation; (g) disrupting focal adhesions; (h) regulating actin polymerization; and (i) treating or inhibiting one or more of intimal hyperplasia, stenosis, restenosis, atherosclerosis, smooth muscle cell tumors, smooth muscle spasm, angina, Prinzmetal's angina (coronary vasospasm), ischemia, stroke, bradycardia, hypertension, pulmonary (lung) hypertension, asthma (bronchospasm), toxemia of pregnancy, pre-term labor, pre-eclampsia/eclampsia, Raynaud's disease or phenomenon, hemolytic-uremia, non-
  • the methods comprise administering to the individual an active HSP20-polypeptide expressing recombinant host cell according to one of the preferred embodiments disclosed above.
  • the subject is a mammal and the recombinant expression vector is of mammalian origin (preferably from the same mammal being treated); in a more preferred embodiment, the individual is a human and the recombinant host cell comprises a recombinant human cell.
  • the nucleic acid encoding the protein kinase A or protein kinase G catalytic subunit is derived from a mammal; preferably a human.
  • treat or “treating” means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorders) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorders).
  • the term “inhibit” or “inhibiting” means to limit the disorder in individuals at risk of developing the disorder.
  • a 1600 bp cDNA encoding the type I catalytic subunit of cGMP-dependent protein kinase from Bos taurus (encoded residues 334-670) was liberated from pBKS+ cPKG (gift from Dr. Thomas Lincoln, University of South Alabama) by Ncol-Xhol digestion and cloned into a NcoI-XhoI-digested shuttle vector pET29a yielding pET29a PKG.
  • pACYC Duet PKG was constructed by ligating the Bglll-Xhol insert of pET29a PKG and the backbone of BgIII- Xhol-digested pACYC Duet (Stratagene).
  • pET14b TAT-HSP20 full length HSP20 conjugated to YGRKKRRQRRR (SEQ ID NO-.37) via a linker
  • pACYC Duet PKG and pET14b TAT-HSP20 were co-transformed into the E. coli expression strain BL21(DE3) and a single colony resistant to 50 mg/L ampicillin and 30 mg/L chloramphenicol used to inoculate 400 mL of Luria Broth (LB) seed culture and grown overnight on a platform shaker (220 rpm at 37 0 C).
  • Frozen cells were thawed and thoroughly resuspended in 100 mL IX TNE buffer (50 mM NaCl 5 1 mM EDTA, and 500 mM Tris-HCl, pH 8.0, 1OnM Caliculin A, 50 ⁇ l Sigma Phosphatase Inhibitor Cocktail II, 50 ⁇ l Sigma Protease Inhibitor Cocktail and 200 ⁇ g deoxyribonuclease) and incubated at 4°C for 30 min. After sonication on ice, the inclusion bodies containing recombinant protein were harvested by centrifugation (19,000 g, 10 min). Metal chelation affinity chromatography was used to purify recombinant rTAT-pHSP20.
  • inclusion bodies were first resuspended in binding buffer (20 mM Na 2 HPO- J , 0.5 M NaCl, 50 mM imidazole, pH 7.4, 8 M urea). The sample was then added to Ni 2+ -charged Chelating Sepharose Fast Flow (Pharmacia Biotech, Peapack, NJ) and incubated for 30 min at room temperature. The resin was then loaded in a water-chilled XK-26 column and washed extensively with binding buffer on an AKTA fast-performance liquid chromatography system (PHARMACIA BIOTECH). Protein was refolded by using an overnight linear gradient of urea from 8 to 0 M and eluted with binding buffer containing 500 mM imidazole.
  • binding buffer 20 mM Na 2 HPO- J , 0.5 M NaCl, 50 mM imidazole, pH 7.4, 8 M urea.
  • the eluate was concentrated and the imidazole removed by repeatedly reducing eluate volume 50% using a stirred ultrafiltration cell (8200, Millipore, Bedford, MA) with a 10,000-nominal molecular weight limit filter under 75 p.s.i. nitrogen and bringing the volume back to 100% again with PSS (physiological salt solution — 140 mM NaCl 5 5 mM KC1 S 1.6 mM CaCl 2 , 1.2 mM MgCl 2 , 1.2 mM Na 2 HPO 4 , 5.6 mM glucose, 2 mM MOPS, and 0.02 mM EDTA, pH 7.4).
  • PSS physiological salt solution — 140 mM NaCl 5 5 mM KC1 S 1.6 mM CaCl 2 , 1.2 mM MgCl 2 , 1.2 mM Na 2 HPO 4 , 5.6 mM glucose, 2 mM MOPS, and 0.02 mM EDTA, pH 7.4
  • Example 3 In vitro analysis of phosphorylation & Co-expression of cPKG and TAT- HSP20
  • the plasmids pET14b-TAT-HSP20 and pACYC-Duet cPKG were constructed as described in Example 1 , sequenced and the proteins expressed in the E. coli strain BL21(DE3), predominantly in insoluble inclusion body forms.
  • One and two dimensional SDS-PAGE, immunoblot analyses and quantification were performed as follows. Protein (75 ⁇ g) in a 120 ⁇ l volume was applied to the entire length of one channel in an isoelectric focusing sample tray (Bio-Rad) to which an immobilized pH gradient strip (linear pH gradient range 3-10, 7-cm length) was applied gel side down.
  • Samples were overlaid with ⁇ 2 ml of mineral oil and allowed to equilibrate for 16 h at room temperature before isoelectric focusing for 20,000 V h. Samples were subsequently equilibrated for 15 min first in an SDS equilibration buffer (50 mM TrisCl pH 8.8, 6 M urea, 30% glycerol, 2% lauryl sulfate, and 0.02% bromophenol blue) containing 1% DTT and second (15 min) in equilibration buffer containing 2.5% iodoacetamide.
  • SDS equilibration buffer 50 mM TrisCl pH 8.8, 6 M urea, 30% glycerol, 2% lauryl sulfate, and 0.02% bromophenol blue
  • samples were separated in the second dimension on precast 4—20% polyacrylamide mini-gels in IX electrophoresis buffer (25 mM TrisCl pH 8.3, 192 mM glycine, 0.1% wt/vol SDS) at 120 V for 1.5 h.
  • IX electrophoresis buffer 25 mM TrisCl pH 8.3, 192 mM glycine, 0.1% wt/vol SDS
  • Electrophoretic transfer of proteins from the gels onto polyvinylidene difluoride membranes was carried out in IX electrophoresis buffer at 50 volts for 12 h at 4°C. The blot was subsequently incubated with the appropriate antibody.
  • Membranes were scanned (Odyssey Infrared Imaging System, Li-Cor Biosciences, Lincoln, NE), and the intensities of selected bands were quantified by using software packaged with the instrument.
  • TAT-pHSP20 is expressed and phosphorylated in E. coli.
  • TAT-pHSP20 was phosphorylated by co-expression with cPKG.
  • purified phosphorylated and non-phosphorylated protein samples were separated by isoelectric focusing followed by SDS-PAGE, blotted to nitrocellulose and probed with anti-HSP20 antibodies.
  • TAT-HSP20 co-expressed with cPKG yields two anti-HSP20 immunoreactive polypeptides.
  • Two-dimensional gel electrophoresis and Western blot analyses of purified recombinant proteins, PTD-HSP27, TAT-HSP20, TAT-pHSP20 was performed.
  • TAT-HSP20 75 ⁇ g of recombinant protein were subjected to isoelectric focusing (linear pH gradient range 3-10, 7 cm length) then separated in the second dimension (4-20% polyacrylamide). Proteins were transferred onto PVDF membranes and immunoblots were incubated with mouse anti-HSP20 and IRDye 700DX conjugated affinity purified goat anti- mouse secondary antibodies. A control protein of weight and composition similar to TAT- HSP20 (PTD-HSP27) did not react with anti-HSP20 antibodies demonstrating antibody specificity. Immunoblots of TAT-HSP20 purified from E. coli revealed a single spot at a pi of ⁇ 9.0 and an electrophoretic mobility of ⁇ 25 kD.
  • Sequences comprising the amino- terminal PTD are basic in nature, can alter electrophoretic mobility and contribute to a higher pi (-9.0) compared to HSP20 without a PTD (pi - 5.9, niass ⁇ 17kD) [6, 13].
  • immunoblots of TAT-pHSP20 purified from E. coli co-expressing cPKG revealed two immunoreactive proteins. One spot exhibited a pi of -9.0 and a mass of 25 kD while the second spot was more acidic by about 0.4 pi units.
  • Sensitive fluorescence-based quantitative analyses of these spots revealed that the more acidic form had a relative intensity of 3.46 (arbitrary units) while the intensity of the more basic spot was 3.70 indicating that the more acidic or phosphorylated spot represented 48% of the total anti-HSP20 immunoreactive protein population isolated from E. coli.
  • Sprague-Dawley rats (40Og) were sacrificed via CO 2 asphyxiation and the aorta of each rat was dissected out, cleaned of excess fat and connective tissue, stored in PSS buffer and 1 hr later cut transversely into rings that were 1 mm in width.
  • Arterial tissues were tied to 4.0 silk, fixed at one end to a stainless steel wire, and attached to a Kent Scientific (Litchfield, CT) force transducer (TRNOOl) interfaced with a Data Translation analog-to-digital board, DT2801 (Data Translation, Marlboro, MA). Data were acquired with Powerlab software (AD Instruments, Colorado Springs, CO).
  • the rings were repeatedly contracted with 110 mM KCl (with equimolar replacement of NaCl in buffer), and the length was progressively adjusted until maximal tension was obtained.
  • the tissue was washed with PSS buffer and equilibrated for at least 30 min between contractions. Contractile responses to serotonin (5- hydroxytryptamine, 5-HT) were subsequently tested by treating the tissue with increasing doses (0.01, 0.1, and l ⁇ M) of 5-HT (not shown). Tissues were rinsed and treated with 0.5 ⁇ M 5-HT (submaximal dose) to determine pretreatment contraction. Any tissue failing to contract was considered nonviable and was not used in further experiments.
  • rTAT-pHSP20 (lO ⁇ M) protein was added to the bath and the ring incubated for 10 min. The effectiveness of the pretreatment was assessed by addition of 0.5 ⁇ M 5-HT, and the contractile response recorded. Percent contraction was calculated relative to force generated with 5-HT prior to pretreatment with rTAT-pHSP20.
  • Protein extracts of vascular rings were obtained by flash freezing tissues still connected to the transducer, then vortexing frozen vascular rings in UDC (9M urea, 2% CHAPS, 10 mM DTT) sample buffer for 1 hr.
  • tissues were contracted with 0.5 ⁇ M 5-HT (submaximal dose) followed by increasing doses of peptide (100 ⁇ M, 500 ⁇ M and 1 mM, respectively).
  • rTAT-pHSP20 The physiological activity of rTAT-pHSP20 from bacterial co-expression experiments was assessed in muscle bath experiments. Rings of male rat aorta were washed, equilibrated with PSS and treated with sub-maximal doses of 5-HT to determine tissue responsiveness. After washing in PSS and letting tension return to baseline levels, rings were incubated in PSS buffer alone or in PSS buffer containing rTAT-pHSP20 (lO ⁇ M for 10 min. Tissues were subsequently challenged with the same sub-maximal doses of 5-HT (0.5 ⁇ M) and the contractile responses recorded. There were no significant differences in the magnitude of contraction in response to serial treatments with 5HT.
  • Peptide mimetics are advantageous in determining full- length protein activity as they have fewer domains available for binding/interaction, are usually easier to synthesize in large quantities, and less susceptible to proteolytic activity.
  • rings of rat aorta contracted with 5-HT displayed steady contractions that were sustained for long periods of time (> 20 min)
  • treatment of aorta rings with increasing amounts of PTD-pHSP20 led to a dose-dependent decrease in contraction that was maximal at a PTD-pHSP20 concentration of 1 mM (data not shown).
  • NIH 3T3 cell culture and Effect of rTAT-pHSP20 on actin stress fibers NIH 3T3 cells (ATCC CRL 1658) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) BCS, penicillin (100 U/ml), and lOO ⁇ g/ml streptomycin (Gibco/BRL), L-glutamine (4 mM) in a CO 2 incubator (5% CO 2 ) at 37 0 C.
  • DMEM Dulbecco's modified Eagle's medium
  • BCS penicillin
  • Gabco/BRL lOO ⁇ g/ml streptomycin
  • Serum free media supplemented with insulin-transfe ⁇ in sodium selenite (5 ng/ml) and HEPES (pH 7.55 - 25mM) was used to quiesce and potentiate cells for stress fiber formation.
  • serum-starved cells seeded on coverslips in 6-well dishes (50-70% confluent) were rinsed briefly with phosphate buffered saline (PBS) then treated with recombinant TAT-pHSP20 (10 ⁇ M) in PSS for 1 h at 37 0 C.
  • PBS phosphate buffered saline
  • Purified recombinant TAT-pHSP20 induces actin rearrangements in 3T3 cells.
  • Cells were then washed with PBS and processed for fluorescence microscopy. Immunofluorescence microscopic images of NIH 3T3 cells serum starved for 24 hrs — treated for one hour with physiological salt solution (PSS) or treated with lO ⁇ M recombinant TAT-pHSP20 recombinant protein in PSS were obtained.
  • Cells were washed, fixed, detergent permeabilized, then probed with Alexa568-phalloidin and DAPI.
  • Cells treated with TAT-pHSP20 lack stress fibers and central actin filaments. Actin filaments are organized evenly throughout each cell and prominent stress fibers are visible.
  • cPKG catalytic subunit of bovine PKG
  • bacterial extracts from cells co-expressing TAT-HSP20 and cPKG as well as refolded TAT-pHSP20 purified under denaturing conditions revealed proteins that were immunoreactive to anti-phospho-HSP20 IgGs.
  • two-dimensional gel electrophoresis of TAT-HSP20 protein obtained from cells co-expressing cPKG, but not TAT-HSP20 alone resulted in the appearance of a second, more acidic, anti-HSP20- immunoreactive spot consistent with TAT-HSP20 phosphorylation.
  • biological assays wherein TAT-pHSP20 was examined for its effects in inhibiting smooth muscle contraction revealed tissue responses indicative of phosphorylation.
  • 14-3-3 proteins comprise a highly conserved family of dimeric and cytosolic proteins that bind other proteins in a phosphorylation-dependent manner and thereby modulate their activity or prevent interaction with other proteins.
  • the intracellular receptor for the peptide analog of HSP20 (PTD-pHSP20) is the scaffolding protein 14-3-3 [10], Consequently, we expected similarities in the cellular responses to full-length rTAT- pHSP20 (described here) and the peptide mimics of HSP20. After either treatment, stress fibers were markedly reduced with most actin redistributed to the cell periphery [10].
  • phosphorylated HSP20 may modulate actin cytoskeletal dynamics by competing with phosphorylated cofilin for binding to 14-3-3. Once released, cofilin is then dephosphorylated, activated and available to sever actin filaments.
  • XP 867488 PREDICTED similar to cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta) isoform 5 [Canis familiaris]
  • XP 867517 PREDICTED similar to cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta) isoform 8 [Canis familiaris]
  • XP 867499 PREDICTED similar to cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta) isoform 6 [Canis familiaris]
  • XP 867524 PREDICTED similar to cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta) isoform 9 [Canis familiaris]
  • P49673 cAMP-dependent protein kinase catalytic subunit P49673 cAMP-dependent protein kinase catalytic subunit (PKA C)
  • XP 867534 PREDICTED similar to cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta) isoform 10 [Canis familiaris]
  • XP 867543 PREDICTED similar to cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta) isoform 11 [Canis familiaris]
  • XP 867507 PREDICTED similar to cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta) isoform 7 [Canis familiaris]
  • P05383 cAMP-dependent protein kinase, beta-catalytic subunit (PKA C-beta)
  • CAI39143 cAMP-dependent protein kinase, catalytic subunit 2-3 [Paramecium tetraurelia]
  • CAG03461 unnamed protein product [Tetraodon nigroviridis]
  • BAE29331 unnamed protein product [Mus musculus]
  • XP 215070 similar to protein kinase, cAMP dependent, catalytic, beta [Rattus norvegicus]
  • BAE24005 unnamed protein product [Mus musculus]
  • AAA30424 cAMP-dependent protein kinase ll-beta catalytic subunit
  • NP 777010 cAMP-dependent protein kinase catalytic subunit beta [Bos taurus]
  • NP 740960 protein KINase family member [Caenorhabditis elegans]
  • JC7968 cyclic adenosine S'. ⁇ '-monophosphate (cAMP)-depe ⁇ dent protein kinase (EC 2.7.1.37). catalytic subunit - rainbow trout

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Abstract

L'invention concerne des cellules hôtes recombinées ainsi que des procédés de fabrication des polypeptides de la protéine de choc thermique 20 (HSP20) phosphorylée ou toute autre cible de la protéine kinase A ou de la protéine kinase G.
PCT/US2007/012320 2006-05-25 2007-05-23 Expression bactérienne de la hsp20 phosphorylée et leurs réactifs Ceased WO2007139846A2 (fr)

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WO2007139846A3 WO2007139846A3 (fr) 2008-06-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008092015A3 (fr) * 2007-01-24 2009-01-29 Univ Arizona MÉTHODES DE TRAITEMENT DES TROUBLES DES MUSCLES LISSES DES VOIES AÉRIENNES CHEZ DES SUJETS DÉSENSIBILISÉS À UN TRAITEMENT PAR AGONISTE DES RÉCEPTEURS β-ADRÉNERGIQUES
WO2022154079A1 (fr) * 2021-01-18 2022-07-21 国立研究開発法人産業技術総合研究所 Procédé d'introduction de protéine dans une cellule animale

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005506836A (ja) * 2001-08-23 2005-03-10 アリゾナ・ボード・オブ・リージェンツ 平滑筋治療用薬剤および方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008092015A3 (fr) * 2007-01-24 2009-01-29 Univ Arizona MÉTHODES DE TRAITEMENT DES TROUBLES DES MUSCLES LISSES DES VOIES AÉRIENNES CHEZ DES SUJETS DÉSENSIBILISÉS À UN TRAITEMENT PAR AGONISTE DES RÉCEPTEURS β-ADRÉNERGIQUES
WO2022154079A1 (fr) * 2021-01-18 2022-07-21 国立研究開発法人産業技術総合研究所 Procédé d'introduction de protéine dans une cellule animale

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