AU2016385177B2 - Novel fusion protein comprising transcription modulation domain of P65 and protein transport domain and use thereof - Google Patents
Novel fusion protein comprising transcription modulation domain of P65 and protein transport domain and use thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Microbiology (AREA)
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a new fusion protein comprising a transcription modulation domain of p65 which is a subunit of the transcription factor NF-κB, and a protein transport domain and a use thereof. The fusion protein according to the present invention has the effects of inhibiting the transcription of NF-κB and IL-2 by competitive inhibition, inhibiting the secretion of inflammatory cytokines by LPS, and inhibiting the production of IL-2, IFN-γ, IL-4, IL-17A and IL-10 in splenocytes, and thus can be useful as a composition for the prevention or treatment of NF-κB hyperactivity-related diseases.
Description
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[Invention Title]
MODULATION DOMAIN OF p65 AND PROTEIN TRANSPORT DOMAIN AND
[Technical Field]
The present invention was made with the support of Future Creation Science
division of Korean Government under a subject No. NRF-2014R1A2A1A10052466,
2009-0083522(ERC), 10044494, and with the support of Yonsei University under a
subject No. 2015-22-0065. The subject No. NRF-2014R1A2A1A10052466 was
conducted under "Middle-Grade Researcher Supporting Project" within the project
named "Development of new material for immunological disease customized for Treg,
which is the control cell that invigorates the immune system" by "Industry-Academic
Cooperation Foundation, YONSEI University" under the management of the National
Research Foundation of Korea, from 1 November, 2014 to 31 October, 2017. The
subject No. 2009-0083522(ERC) was conducted under "National Research Foundation
of Korea- SRC(Science Research Center)/ERC(Engineering Research Center)" within
the project named "ERC/Center for Research on Fulfillment of Protein Function
Control (3/3, level 2)" by "Industry-Academic Cooperation Foundation, YONSEI
University" under the management of the National Research Foundation of Korea,
from 1 September, 2009 to 29 February, 2016. The subject No. 10044494 was
conducted under "SW Computing Industry source technology development project"
within the project named "WiseKB: Development of Knowledge Base in Self-study
Form Grounded on an Understanding of Bigdata and Deductive Technology" by
!5 "Saltlux Inc." under the management of the Institute for Information & communications Technology Promotion, from 1 July, 2013 to 28 February, 2016. The subject No. 2015-22-0065 was conducted under "Support for research within the university" within the project named "[Pioneers of the Future - International]
Development of Individually-customized New Medicine made out of Protein, for the
Purpose of Controlling the Function of the Transcription Factor that has the Ability to
Control Gene Expression of a Subset of T cell which is a Morfibic factor
for Autoimmune Disease" by " Industry-Academic Cooperation Foundation, YONSEI
University" under the management of the Yonsei University, from 1 September 2015
to 31 August, 2016. The present invention relates to a novel fusion protein
comprising the transcription modulation domain (TMD) of the transcription factor NF
KB subunit p65 and a protein transduction domain (PTD) and to the use thereof.
[Background Art]
Cytokines that are regulated by the transcription factor NF-KB include tumor
necrosis factor-a (TNF-a), interleukin-i (IL-1), interleukin-6 and granulocyte
macrophage colony stimulating factor (GM-CSF), and chemokines that are regulated by
NF-KB include interleukin-8, macrophage-inflammatory protein-la (MIP-1a), methyl
accepting chemotaxis protein-i (MCP-1) and eotaxin. In addition, adhesion molecules
that are regulated by NF-KB include E-selectin, vascular cell adhesion molecule-i
(VCAM-1), endothelial leukocyte adhesion molecule-i (ELAM-1) and intercellular cell
adhesion molecule-i (ICAM-1), and inducible enzymes that are regulated by NF-KB
include cyclooxygenase-2 (COX-2) and the like. Thus, NF-KB is involved in almost all
physiological reactions in the body.
The transcription factor NF-KB is composed of different subunits composed of
homodimers or heterodimers. NF-KB proteins include RelA (p65), c-Rel, Rel-B, NF
KBI (p50) and NF-KB2 (p52), and p50 and p52 are produced from NF-KBI (p105) and
NF-KB2 (p100), respectively, which are their precursors. The NF-KB proteins each
contains about 300 amino acids, called the N-terminal Rel-homology domain (RHD),
which is involved in dimerization, binding to specific DNA and reactions with IKB
protein. Furthermore, these proteins also contain a nuclear localization signal (NLS)
that enables the proteins to act as transcription factors by localization to the nucleus. In
addition, the NF-KB proteins may be classified, according to the presence or absence of
the C-terminal transactivation domain (TAD), into class I (NF-KB1 and NF-KB2) and
class II (RelA/p65, RelB, and c-Rel). Class II has a transactivation domain that enables
the NF-KB proteins to act as transcription factors without needing other NF-B domains,
and class I has no transactivation domain. Dimers are formed between the NF-KB
proteins of the two classes to act as DNA transcription factors. Among the dimers, a
p50/p65 dimer is most frequently present. RelA(p65), RelB and c-Rel have a C
terminal transactivation domain, and thus can activate the expression of their target genes.
On the contrary, p50 and p52, which are NF-B1 and NF-KB2, respectively, have no C
terminal transactivation domain, and thus homodimers of p50 and p52 do not act as
transcription factors if they do not have bound thereto proteins such as co-activators
having a transactivation domain.
A protein transduction domain (PTD) is a short peptide having strong
hydrophobicity, and is known to effectively transduce physiologically active molecules
such as proteins, DNA and RNA, fused therewith, into cells. The present inventors
have developed two PTDs to date, and the PTDs are disclosed in detail in WO
2003059940 and WO 2003059941. Because the protein transduction domain can
transduce a physiologically active molecule not only into the cytoplasm but also into the
nucleus, it has a property suitable for transducing a modified transcription factor, which
is the key substance of the present invention, into the nucleus.
Therefore, the present inventors have attempted to use a fusion protein
comprising the transduction modulation domain of the NF-KB subunit p65 and a protein
transduction domain to inhibit the transcription and activity of NF-KB by competitive
inhibition to thereby treat effectively a disease caused by NF-B overactivity.
Throughout the specification, a number of publications and patent documents are
referred to and cited. The disclosure of the cited publications and patent documents is
incorporated herein by reference in its entirety to more clearly describe the state of the
related art and the present invention.
[Disclosure]
D [Technical Problem]
The present inventors have made extensive efforts to develop a substance
capable of effectively preventing, alleviating or treating a disease caused by the
overactivity of NF-KB. As a result, the present inventors have found that, when a fusion
protein comprising the transcription modulation domain of the NF-KB subunit p65 and a
protein transduction domain is used, it can inhibit the transcription and activity of NF-KB
by competitive inhibition, thereby completing the present invention.
Therefore, it is an aspect of the present invention to provide a fusion protein
comprising the transcription modulation domain of the NF-KB subunit p65 and a protein
transduction domain.
Another aspect of the present invention is to provide an inhibitor of NF-KB
transcription or activity, comprising the fusion protein of the present invention.
Still another aspect of the present invention is to provide a pharmaceutical
composition for the prevention or treatment of a disease related to NF-B overactivity,
the pharmaceutical composition comprising the fusion protein of the present invention as
an active ingredient.
Still another aspect of the present invention is to provide a method for inhibiting
transcription or activity of NF-B, and a method of preventing, improving or treating a
disease related to NF-KB overactivity.
Other aspect and advantages of the present invention will become more apparent
from the following detailed description, the appended claims and the accompanying
drawings.
[Technical Solution]
In accordance with one aspect of the present invention, there is provided a fusion
D protein comprising the transcription modulation domain of the NF-KB subunit p65 and a
protein transduction domain. The fusion protein of the present invention inhibits NF-KB
transcription by competitive inhibition.
The present inventors have made extensive efforts to develop a substance
capable of effectively preventing, alleviating or treating a disease caused by NF-KB
overactivity. As a result, the present inventors have found that, when a fusion protein
comprising the transcription modulation domain of the NF-KB subunit p65 and a protein
transduction domain is used, it can inhibit the transcription and activity of NF-KB by
competitive inhibition.
As used herein, the term "transcription modulation domain" means a portion of a
transcription factor, which is composed only of a DNA binding domain without a
transactivation domain. As demonstrated in the examples below, the fusion protein of
the present invention has no transactivation domain, but has a DNA binding domain, and
thus it can bind to a target promoter, but cannot promote transcription. Accordingly, the
fusion protein of the present invention is a dominant negative mutant for the NF-KB gene,
!5 and thus can act as a competitive inhibitor against wild-type NF-KB in cells to inhibit the transcription and activity of NF-KB.
In one embodiment, NF-B that is used in the present invention is NF-KB
selected from the group consisting of RelA (p65), c-Rel, Rel-B, NF-KB1 (p50) and NF
KB2 (p52). In a specific embodiment, NF-B that is used in the present invention is
RelA (p65).
In one embodiment, the transcription modulation domain of NF-KB that is used
in the present invention comprises an amino acid sequence of SEQ ID NO: 1. In
another embodiment of the present invention, the transcription modulation domain of
NF-B, which comprises the amino acid sequence of SEQ ID NO: 1, is encoded by a
nucleotide sequence of SEQ ID NO: 3.
As used herein, the term "protein transduction domain" is a short, strongly
hydrophobic peptide consisting of 7-50 amino acids, and means a domain capable of
transducing not only a protein having a molecular weight of 120 kDa or more, but also
DNA or RNA, into cells. As demonstrated in the examples below, it could be seen that,
unlike the fusion protein of the present invention, a protein to which the protein
transduction domain of the present invention is not attached (that is, p65-TMD composed
only of the transcription modulation domain) has no effects on the inhibition of
transcription of NF-KB and IL-2, the inhibition of LPS-induced secretion of
inflammatory cytokines, and the inhibition of production of IL-2, IFN-y, IL-4, IL-17A
and IL-10 in splenocytes.
According to one embodiment of the present invention, the protein transduction
domain that is used in the present invention is selected from the group consisting of Hph
1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-i (peptide-1), PTD-5 (protein
transduction domain-5), 7R, 9R, 1IR and CTP (cytoplamic transduction peptide). As
used herein, the terms "7R", "9R" and "11" mean peptides consisting of 7, 9 and 11 arginine residues, respectively. According to a specific embodiment, the protein transduction domain that is used in the present invention is Hph-1.
According to one embodiment of the present invention, the protein transduction
domain that is used in the present invention comprises an amino acid sequence of SEQ
ID NO: 2. According to another embodiment of the present invention, the protein
transduction domain comprising the amino acid sequence of SEQ ID NO: 2 according to
the present invention is encoded by a nucleotide sequence of SEQ ID NO: 4.
According to one embodiment of the present invention, the fusion protein of the
present invention comprises an amino acid sequence of SEQ ID NO: 5. According to a
specific embodiment of the present invention, a fusion protein comprising an amino acid
sequence of SEQ ID NO: 5 is encoded by a nucleotide sequence of SEQ ID NO: 6.
In accordance with another aspect of the present invention, there is provided an
inhibitor of NF-KB transcription or activity, comprising the fusion protein of the present
invention. As demonstrated in the examples below, it was shown that the fusion protein
of the present invention could inhibit the transcription and activity of NF-KB and IL-2
(FIG. 6), inhibit the LPS-induced secretion of inflammatory cytokines (FIG. 7), and
inhibit the production of IL-2, IFN-y, IL-4, IL-I7A and IL-10 which are expressed by T
cell activation stimulated by anti-CD3/CD28 in splenocytes (FIG. 9), indicating that the
fusion protein can inhibit the NF-KB-induced proliferation and differentiation of T cells
and the NF-KB-induced secretion of inflammatory cytokines. This suggests that the
fusion protein of the present invention can effectively prevent, alleviate or treat a disease
caused by the overactivity of NF-KB.
In accordance with another aspect of the present invention, there is provided a
pharmaceutical composition for the prevention or treatment of a disease related to NF-KB
overactivity, the pharmaceutical composition comprising the fusion protein of the present invention as an active ingredient.
As used herein, the term "treatment" refers to: (a) suppressing the progression of
disease, disorder, or symptom; (b) reducing disease, disorder, or symptom; or (c) curing
the disease, disorder, or symptom. The composition of the present invention suppresses
the development of symptoms of metabolic disease, or removes or reduces the symptoms
of metabolic disease. Therefore, the composition of the present invention may be a
composition for treating disease related to NF-KB overactivity, or may be applied as a
treatment adjuvant for the disease when administered with other composition for treating
a disease related to NF-KB overactivity. As used herein, the term "treatment" or
"treatment agent" includes a meaning of "treatment aid" or "therapeutic adjuvant".
As demonstrated in the examples below, the fusion protein of the present
invention inhibits the transcription of NF-KB and IL-2, inhibits the LPS-induced
secretion of inflammatory cytokines, and inhibits the production of IL-2, IFN-y, IL-4, IL
17AandIL-10 insplenocytes. Thus, the fusion protein of the present invention maybe
effectively used as a composition for efficiently preventing or treating various diseases
related to NF-KB overactivity.
In one embodiment of the present invention, the disease related to NF-KB
overactivity in the present invention is an inflammatory disease or an autoimmune
disease. In another embodiment of the present invention, the disease related to NF-KB
overactivity in the present invention is a disease selected from the group consisting of
septic shock, allergic asthma, allergic nasitis, atopic dermatitis, systemic lupus
erythematosus, rheumatoid arthritis, ulcerative colitis, dacryoadenitis, Alzheimer's
disease, stroke, arteriosclerosis, vascular restenosis, type I diabetes, type II diabetes,
urticaria, conjunctivitis, psoriasis, systemic inflammatory response syndrome,
polymyositis, dermatomyositis, polyarthritis nodosa, mixed connective tissue disease,
Sjogren's syndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis, diabetic
retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, Celiac disease,
myasthenia gravis, pemphigus vulgaris, viral diseases, bacterial diseases, radiation
induced disorders, arteriosclerosis, hemangioma, angiofibroma, reperfusion injury, and
cardiac hypertrophy. According to a specific embodiment, the disease related to NF-KB
overactivity in the present invention is septic shock. Septic shock that causes a severe
systemic inflammatory reaction due to microbial infection shows abnormalities in the
cardiovascular system and vasomotor factors by endotoxins and inflammatory mediators
circulating the blood vessels. For this reason, hypovolemia occurs due to dehydration
and leakage of intravascular liquid, and vascular contraction and relaxation in capillary
vessels occur to cause abnormalities in blood supply, and vasculitis and thrombosis make
tissue reflux more difficult, resulting in tissue hypoxia and metabolic acidosis. In
particular, endotoxins activate NF-KB in immunocytes (macrophages, granulocytes,
dendritic cells, and lymphocytes) to secrete cytokines such as tumor necrosis factor,
linterleukin-1 and linterleukin-6, and such cytokines stimulate neutrophils, endothelial
cells, platelets or inflammatory mediator-releasing cells to cause systemic reactions. As
demonstrated in the examples below, it was shown that the fusion protein of the present
invention inhibited the secretion of inflammatory cytokines in septic shock models
induced by LPS and increased the survival rate of the models (FIG. 10).
Thus, it can be seen that the fusion protein of the present invention is effective
for the prevention or treatment of a disease related to NF-B overactivity.
If the composition of the present invention is prepared as a pharmaceutical
composition, the pharmaceutical composition of the present invention contains a
pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable
carrier that is contained in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations. The pharmaceutical composition may further contain, in addition to the above-described components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative or the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's PharmaceuticalSciences ( 19 th ed., 1995).
The pharmaceutical composition of the present invention may be administered
orally or parenterally. When the pharmaceutical composition is administered
parenterally, it may be injected intravenously, subcutaneously, intramuscularly,
intraperitoneally, transdermally, or the like. In one embodiment of the present invention,
the pharmaceutical composition of the present invention may be administered by
intraperitoneal injection.
The suitable dose of the pharmaceutical composition of the present invention
may vary depending on various factors, including the formulation method, the mode of
administration, the patient's age, weight, sex, disease condition and diet, the time of
administration, the route of administration, the rate of excretion, and reaction sensitivity.
The daily dose of the pharmaceutical composition according to the present invention is,
for example, 0.0001-1000 mg/kg.
The pharmaceutical composition of the present invention may be formulated
with a pharmaceutically acceptable carrier and/or excipient in a unit dosage form or a
multiple dosage form, according to a method that can be easily carried out by a skilled person in the art to which the present invention pertains. Herein, the formulation may be a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further contain a dispersing agent or a stabilizing agent.
Because the NF-KB transcription or activity inhibitor of the present invention and
the composition for the prevention or treatment of a disease related to NF-B overactivity
according to the present invention commonly comprise the above-described fusion
protein, the description of the contents that are common in relation to the fusion protein
is omitted in order to avoid excessive complexity of description.
In accordance with another aspect of the present invention, there is provided a
method of inhibiting transcription or activity of NF-KB, the method comprising
administering a composition comprising the fusion protein of the present invention as an
active ingredient.
In accordance with another aspect of the present invention, there is provided a
method of preventing, improving or treating a disease related to NF-KB overactivity, the
method comprising administering, to a subject in need, a composition comprising the
fusion protein of the present invention as an active ingredient.
As used herein, the term "administration" or "administer" refers to a method
wherein a therapeutically effective amount of the composition of the present invention is
directly administered to a subject to form the same amount thereof in the body of the
subject. Therefore, the term "administer" includes the injection of an active ingredient
(the fusion protein of the present invention) around a site of lesion, and thus the term
"administer" is used in the same meaning as the term "inject".
The term "therapeutically effective amount" of the composition refers to the
content, which is sufficient to provide a therapeutic or prophylactic effect to a subject to
be administered, and thus the term has a meaning including "prophylactically effective
amount". As used herein, the term "subject" includes, but is not limited to, human,
mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, beaver, or rhesus
monkey. Specifically, the subject of the present invention is human.
Since the method of inhibiting the transcription or activity NF-KB, and the
method for preventing, improving or treating the disease related to NF-KB overactivity
commonly comprise the above-described fusion protein and the use thereof, descriptions
D of overlapping contents therebetween are omitted to avoid excessive complication of the
specification due to repeated descriptions thereof.
Any discussion of documents, acts, materials, devices, articles or the like which
has been included in the present specification is not to be taken as an admission that any
or all of these matters form part of the prior art base or were common general knowledge
in the field relevant to the present disclosure as it existed before the priority date of each
of the appended claims.
Throughout this specification the word "comprise", or variations such as
"comprises" or "comprising", will be understood to imply the inclusion of a stated
element, integer or step, or group of elements, integers or steps, but not the exclusion of
any other element, integer or step, or group of elements, integers or steps.
[Advantageous Effects]
The features and advantages of the present invention are as follows.
(a) The present invention provides a fusion protein comprising the transcription
!5 modulation domain of the NF-KB subunit p65 and a protein transduction domain, and the use thereof.
(b) The fusion protein of the present invention has the effects of inhibiting the
transcription of NF-B and IL-2 by competitive inhibition and inhibiting the LPS
induced secretion of inflammatory cytokines and also inhibiting the production of IL-2,
IFN-y, IL-4, IL-17A and IL-10 in splenocytes, and thus can be effectively used as a
composition for the prevention or treatment of a disease related to NF-B overactivity.
[Description of Drawings]
FIG. 1 shows the structure of a fusion protein (nt-p65-TMD) of a p65
transcription modulation domain (p65-TMD) and Hph-1 (PTD) according to an
12a embodiment of the present invention. DBD: DNA binding domain; IBD: IKB binding domain; NLS: nuclear localization sequence; TAD: transactivation domain.
FIG. 2 shows the results of Coomassie blue staining of the fusion protein (nt
p65-TMD) according to the present invention.
FIG. 3 shows the results of transduction of the fusion protein (nt-p65-TMD) of
the present invention into BV2 and Jurkat T cells.
FIG. 4 shows the results of transduction of the fusion protein (nt-p65-TMD) of
the present invention into the nuclei of BV2 and HeLa cells.
FIG. 5 shows the results of cytotoxicity of the fusion protein (nt-p65-TMD) of
the present invention in BV2 and spleen cells.
FIG. 6 shows the effect of the fusion protein (nt-p65-TMD) of the present
invention on competitive inhibition of transcription.
FIG. 7 shows the effect of the fusion protein (nt-p65-TMD) of the present
invention on the inhibition of LPS-induced expression of TNF-a, IL- Iand IL-6.
FIG. 8a shows results indicating that the fusion protein (nt-p65-TMD) of the
present invention specifically inhibits NF-KB transcription induced by T-cell activation
stimulated by anti-CD3/CD28. FIG. 8b show results indicating that fusion protein (nt
p65-TMD) of the present invention does not influence signal transduction pathways
induced by T-cell activation stimulated by anti-CD3/CD28.
FIG. 9 shows results indicating that the fusion protein (nt-p65-TMD) of the
present invention inhibits the expression of IL-2, IFN-y, IL-4, IL-17A and IL-10, induced
by T-cell activation stimulated by anti-CD3/CD28.
FIG. 10 shows results indicating that the fusion protein (nt-p65-TMD) of the
present invention inhibits the secretion of inflammatory cytokines (TNF-a, IL- IPand IL
6) in septic shock animal models to thereby increase the survival rate of the animal models.
[Best Mode]
Hereinafter, the present invention will be described in further detail with
reference to examples. It will be obvious to those skilled in the art that these examples
are for illustrative purposes and are not intended to limit the scope of the present
invention.
Examples
Example 1: Preparation of a Recombinant Fusion Protein Comprising p65
Transcription Modulation Domain and Protein Transduction Domain
The protein transduction domain (PTD) Hph-1 (SEQ ID NO: 4) was cloned into
the p65 transcription modulation domain p65-TMD (SEQ ID NO: 2) and a pET-28a(+)
vector (Novagen) to thereby construct a recombinant fusion DNA (nt-p65-TMD) (SEQ
ID NO: 6). The recombinant fusion DNA was transformed into a BL21
CodonPlus(DE3)-RIPL E. coli strain (Invitrogen). The transformed strain was cultured,
and then treated with 1 mM IPTG (isopropyl-p-D-thiogalactopyranoside, Duchefa), after
which protein expression in the strain was induced at 37°C for 5 hours. Next, only the
cells were harvested, lysed with lysis buffer (10 mM imidazole, 50 mM NaH 2PO 4 , 300
mM NaCl, pH 8.0), and then disrupted with a homogenizer. The fusion protein was
bound to Ni-NTA beads (Qiagen) by 6 histidine residues linked to the upstream region of
the protein. The protein was loaded onto HisTrap chromatography columns (Bio-Rad),
and washed sufficiently with washing buffer (30 mM imidazole, 50 mM NaH 2PO 4 , 300
mM NaCl, pH 8.0), and the protein was eluted with elution buffer (250 mM imidazole,
50 mM NaH 2 PO 4, 300 mM NaCl and pH 8.0). Using PD-10 Sephadex G-25 (GE
Healthcare), imidazole and NaCl were removed while the buffer was replaced with 10%
!5 glycerol-containing PBS. Because the obtained protein contained endotoxins such as
LPS, it was purified once more using SP beads (SP Sepharose TM Fast Flow, GE
Healthcare) to remove the endotoxins. The resulting protein was bound using binding
buffer (50 mM NaH 2PO 4 , 300 mM NaCl, pH 6.0), and then loaded onto a column and
eluted with elution buffer (50 mM NaH 2PO 4 , 2 M NaCl, pH 6.0). Finally, NaCl was
removed using PD-10 Sephadex G-25, and the buffer was placed with 10% glycerol
containing PBS, after which the resulting protein (SEQ ID NO: 5, recombinant fusion
protein) was stored at 80°C until use in experiments (see FIGS. 1 and 2).
Example 2: Analysis of Intracellular Transduction of nt-p65-TMD
Recombinant Fusion Protein
2-1: Analysis of Intracellular Transduction by Western Blotting
BV2 microglial cells and Jurkat T cells were incubated with the recombinant
fusion protein (nt-p65-TMD) at varying protein concentrations (0, 0.1, 0.5, 1, 2 and 5
pM) or for varying times (0, 1, 2, 4, 6, 12, 24 and 48 hours), and whether or not the
fusion protein was transduced was analyzed by Western blotting.
As a result, it was shown that the fusion protein was well transduced in
proportion to the concentration thereof and was continuously transduced even at 48 hours
while the protein in the cell culture maintained its structure (see FIG. 3).
2-2: Analysis of Whether or Not Fusion Protein is Transduced to Nucleus of
Cells, by Use of Antibody
5 M of the recombinant fusion protein (nt-p65-TMD) of Example 1 was
incubated with BV2 microglial cells and HeLa cells for 1 hour and washed with PBS,
after which the cells were treated with 0.2% Triton X-100 (Sigma-Aldrich) to form an
opening, and a fluorescence-labeled antibody was bound to the recombinant fusion
protein through the opening. Next, the nucleus of the cells was stained with DAPI dye
(Invitrogen), and then the location of fluorescence was determined by a fluorescence microscope to thereby determine the location to which the recombinant fusion protein was transduced.
As a result, it was shown that the fusion protein was well transduced to the
nucleus of the cells by the property of the PTD (see FIG. 4).
Example 3: Analysis of Cytotoxicity of nt-p65-TMD Recombinant Fusion
Protein
In order to confirm that the protein obtained from the E. coli strain by expression
is completely free of LPS so that it is not toxic to cells or animals, a cytotoxicity test was
performed. Varying concentrations of the protein were transduced into BV2 microglial
cells and splenocytes, and then the cells were incubated with WST-8, a substrate that
develops color by dehydrogenase present in living cells.
As a result, it could be seen that the cells treated with the fusion protein showed
no cytotoxicity regardless of the concentration of the fusion protein, unlike cells not
treated with the protein (FIG. 5).
Example 4: Confirmation of the Effect of nt-p65-TMD Recombinant Fusion
Protein on Specific Inhibition of Transcription Factor
4-1: Confirmation of Inhibitory Effect against Transcription of NF-KB and
IL-2 in HEK293T Cells
To directly confirm whether or not the nt-p65-TMD recombinant fusion protein
of Example 1 binds to the promoters of the NF-KB and IL-2 cytokine genes in place of
wild-type p65 to inhibit the expression of the genes, luciferase reporter gene was used.
First, each of NF-KB and IL-2 promoters and wild-type p65 gene, which have luciferase
in the downstream region, was transfected into the nucleus of HEK293T cells, and then
the cells were treated with the nt-p65-TMD recombinant fusion protein.
As a result, it could be seen that, when the cells were treated with the recombinant fusion protein, the expression of luciferase in the cells was effectively inhibited. This suggests that nt-p65-TMD acted as a competitive inhibitor of wild-type p65 to block the p65-binding site of each of the NF-KB and IL-2 promoters (see FIG. 6).
4-2: Confirmation of Inhibitory Effect against Secretion of Inflammatory
Cytokines in BV2 Microglial Cells
BV2 microglial cells were treated with the nt-p65-TMD recombinant fusion
protein of Example 1, and after 1 hour, the cells were treated and incubated with LPS (1
ptg/ml, E. coli serotype 055:B5, Sigma-Aldrich) for 24 hours.
As a result, it could be seen that the expression of TNF-a, IL-IPand IL-6, which
are activated and expressed by LPS, was inhibited by the nt-p65-TMD recombinant
fusion protein (see FIG. 7).
4-3: Confirmation of Specific Inhibitory Effect against NF-KB Transcription
Factor in Jurkat T Cells
Whether or not the nt-p65-TMD recombinant fusion protein of Example 1
specifically inhibits the transcription of NF-KB activated by Jurkat T-cell activation
stimulated by anti-CD3 (1 g/ml, BD Pharmingen) and anti-CD28 (1 g/ml, BD
Pharmingen) was examined. When T cells are activated, not only NF-KB transcription
but also NFAT transcription is activated. For this reason, if the nt-p65-TMD fusion
protein inhibits only NF-KB transcription without influencing NFAT transcription, it can
be seen that the nt-p65-TMD recombinant fusion protein acts as a specific competitive
inhibitor of NF-KB. To confirm this fact, luciferase reporter gene was used. First, NF
KB and NFAT reporter genes having luciferase in the downstream region were
transfected into the nucleus of Jurkat T cells by elctroporation, and then the Jurkat T cells
were stimulated with anti-CD3 and anti-CD28 and treated with the nt-p65-TMD
!5 recombinant fusion protein.
As a result, it could be seen that, when the NF-B-transfected cells were treated
with the recombinant fusion protein, the expression of luciferase in the cells was
effectively inhibited, but NFAT was not influenced by the recombinant fusion protein
(see FIG. 8a).
4-4: Phosphorylation of Intracellular Signal Transduction Protein
In order to examine whether or not the nt-p65-TMD recombinant fusion protein
of Example 1 is involved in tyrosine phosphorylation of proteins related to various
intracellular signal transduction systems, Western blot analysis was performed. Jurkat
T cells were treated with 2 M of nt-p65-TMD for 1 hour, and then stimulated with anti
CD3 (2.5 g/ml) and anti-CD28 (2.5 [g/ml), and whether or not tyrosine phosphorylation
of ZAP-70, p38, JNK or ERK occurred was observed.
As a result, it could be seen that nt-p65-TMD did not influence phosphorylation
of these proteins (see FIG. 8b).
4-5: Inhibition of Production of Cytokines (IL-2, IFN-y, IL-4, IL-17A and
IL-10) in Splenocytes
Splenocytes isolated from the spleens of 6-8-week-old female C57BL/6 mice
were treated with the nt-p65-TMD recombinant fusion protein of Example 1 for 1 hour to
transduce the recombinant fusion protein into the cells. The cells were stimulated with
anti-CD3 (1 g/ml) and anti-CD28 (1 g/ml), and then incubated for 72 hours. Next,
the amounts of cytokines present in the medium were measured by ELISA.
As a result, it could be seen that, when the cells were treated with nt-p65-TMD,
the expression levels of IL-2, IFN-y, IL-4, IL-17A and IL-10 in the cells were greatly
inhibited. In addition, the expression level of CD69, which is indicative of T cell
activation, was measured by FACS Calibur (BD Biosciences), and as a result, it could be
seen that nt-p65-TMD did not influence the expression of CD69 in T cells (FIG. 9).
Example 5: Evaluation of Therapeutic Effect of nt-p65-TMD in Septic
Shock Animal Models
LPS (20 mg/kg) was injected intraperitoneally into 6-8-week-old male BALB/c
mice to make septic shock animal models. After 2 hours and 14 hours, the nt-p65-TMD
recombinant fusion protein of Example 1 was injected intraperitoneally into the animal
models, followed by observation for 6 days.
As a result, it could be seen that, when the animal models were treated with nt
p65-TMD, the secretion of inflammatory cytokines (TNF-a, IL-1j and IL-6) was
inhibited to thereby greatly increase the survival rate of the animal models (see FIG. 10).
Example 6: Acute Toxicity Test for Recombinant Fusion Protein
Using 6-week-old specific pathogen-free (SPF) rats obtained from the Daehan
Experiment Supply Center, an acute toxicity test was performed in the following manner.
The recombinant fusion protein of Example 1 was administered orally once to each
animal group (consisting of two animals) at a dose of 1 g/kg, and then the death, clinical
symptoms and weight changes of the animals were observed, and hematological tests and
blood biochemical tests were performed. In addition, the animals were autopsied, and
whether or not the abdominal organs and the thoracic organs were abnormal was visually
observed.
As a result, in all the animals administered with the test substance, specific
clinical symptoms or dead animals were not found, and in the weight change
measurement, hematological tests, blood biochemical tests and autopsy findings, no
change in toxicity was observed. Thus, it could be seen that the recombinant fusion
protein of the present invention showed no toxicity in the rats even at a dose of 1 g/kg
and that the minimum lethal dose (LD5 o) upon oral administration thereof was 1 g/kg or
more, suggesting that it is a safe substance.
References
Park et al., Intranuclear interactomic inhibition of NF-kB suppresses LPS
induced severe sepsis, Biochemical and Biophysical Research Communications, 2015;
464:711-717.
Although the present disclosure has been described in detail with reference to
the specific features, it will be apparent to those skilled in the art that this description is
only of a preferred embodiment thereof, and does not limit the scope of the present
invention. Thus, the substantial scope of the present invention will be defined by the
appended claims and equivalents thereof.
Claims (16)
1. A fusion protein comprising a transcription modulation domain of NF-KB
and a protein transduction domain, wherein the fusion protein inhibits NF-KB transcription by
competitive inhibition,
wherein the transcription modulation domain of NF-KB consists of an amino acid
sequence of SEQ ID NO: 1.
2. The fusion protein of claim 1, wherein the transcription modulation domain
of NF-KB is encoded by a nucleotide sequence of SEQ ID NO: 2.
3. The fusion protein of claim 1, wherein the protein transduction domain is
selected from the group consisting of Hph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep
(peptide-1), PTD-5 (protein transduction domain-5), 7R, 9R, 11R and CTP (cytoplasmic
transduction peptide).
4. The fusion protein of claim 1, wherein the protein transduction domain is
Hph-1.
5. The fusion protein of claim 1, wherein the protein transduction domain
comprises an amino acid sequence of SEQ ID NO: 3.
6. The fusion protein of claim 5, wherein the protein transduction domain is
encoded by a nucleotide sequence of SEQ ID NO: 4.
7. The fusion protein of claim 1, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO: 5.
8. The fusion protein of claim 7, wherein the fusion protein is encoded by a
nucleotide sequence of SEQ ID NO: 6.
9. An inhibitor of NF-KB transcription or activity, comprising the fusion
protein of claim 1.
10. Use of the fusion protein of claim 1 in the manufacture of a medicament for
prevention or treatment of a disease related to NF-B overactivity.
11. The use of claim 10, wherein the disease related to NF-KB overactivity is an
inflammatory disease or an autoimmune disease.
12. The use of claim 10, wherein the disease related to NF-KB overactivity is a
disease selected from the group consisting of septic shock, allergic asthma, allergic nasitis,
atopic dermatitis, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis,
dacryoadenitis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I
diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis, systemic inflammatory response
syndrome, polymyositis, dermatomyositis, polyarthritis nodosa, mixed connective tissue
disease, Sjogren's syndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis, diabetic
retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, Celiac disease,
myasthenia gravis, pemphigus vulgaris, viral diseases, bacterial diseases, radiation-induced
disorders, arteriosclerosis, hemangioma, angiofibroma, reperfusion injury, and cardiac
hypertrophy.
13. A method for inhibiting transcription or activity of NF- KB, the method
comprising administering a composition comprising the fusion protein of claim 1 as an active
ingredient.
14. A method of preventing, improving or treating a disease related to NF-KB
overactivity, the method comprising administering, to a subject in need, a composition
comprising the fusion protein of claim 1 as an active ingredient.
15. The method of claim 14, wherein the disease related to NF-KB overactivity is
an inflammatory disease or an autoimmune disease.
16. The method of claim 14, wherein the disease related to NF-KB overactivity is
a disease selected from the group consisting of septic shock, allergic asthma, allergic nasitis,
atopic dermatitis, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis,
dacryoadenitis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I
diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis, systemic inflammatory response
syndrome, polymyositis, dermatomyositis, polyarthritis nodosa, mixed connective tissue
disease, Sjogren's syndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis, diabetic
retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, Celiac disease,
myasthenia gravis, pemphigus vulgaris, viral diseases, bacterial diseases, radiation-induced
disorders, arteriosclerosis, hemangioma, angiofibroma, reperfusion injury, and cardiac
hypertrophy.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/KR2016/000104 WO2017119521A1 (en) | 2016-01-06 | 2016-01-06 | Novel fusion protein comprising transcription modulation domain of p65 and protein transport domain and use thereof |
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| AU2016385177B2 true AU2016385177B2 (en) | 2023-10-19 |
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| AU (1) | AU2016385177B2 (en) |
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|---|---|---|---|---|
| KR20120017913A (en) * | 2010-08-20 | 2012-02-29 | 연세대학교 산학협력단 | A inhibitor for transcription factor of fusion protein comprising dna binding domain of transcription factor and protein transduction domain |
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| ATE478676T1 (en) * | 2005-11-04 | 2010-09-15 | Forhumantech Co Ltd | METHOD FOR DELIVERING FUSION POLYPEPTIDE INTO A CELL |
| WO2012023838A2 (en) * | 2010-08-20 | 2012-02-23 | Lee Sang-Kyou | Fusion protein having transcription factor transactivation-regulating domain and protein transduction domain, and transcription factor function inhibitor comprising the same |
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2016
- 2016-01-06 CA CA3010811A patent/CA3010811A1/en active Pending
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|---|---|---|---|---|
| KR20120017913A (en) * | 2010-08-20 | 2012-02-29 | 연세대학교 산학협력단 | A inhibitor for transcription factor of fusion protein comprising dna binding domain of transcription factor and protein transduction domain |
Non-Patent Citations (1)
| Title |
|---|
| PARK, Sung-Dong et al., "Intranuclear interactomic inhibition of NF-κB Suppresses LPS--induced Severe Sepsis'', Biochemical and Biophysical Research Communications. 2015, Vol. 464, pages 711-717 * |
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| JP7051687B2 (en) | 2022-04-11 |
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