DE10109532C1 - New fusion protein that blocks Nef protein, useful for treatment or diagnosis of acquired immune deficiency syndrome, has high specificity and affinity - Google Patents

Abstract

Fusion protein (FP) for blocking the Nef protein of human immune deficiency virus (HIV) comprises: (i) protein domain (PD1) that binds to a di-leucine (LL) motif; (ii) a protein domain (PD2) that binds to a PxxP motif; and (iii) a polypeptide linker between PD1 and 2. Independent claims are also included for the following: (1) nucleic acid sequence (I) that encodes FP; (2) expression vector containing (I); (3) host cells containing the vector of (2); and (4) method for producing FP by culturing cells of (3).

Description

This invention relates to a novel fusion protein for Blocking the HIV-Nef Protein. The invention relates continue nucleic acid sequences that this fusion protein or encode a biologically active part thereof, the Complementary sequence, as well as an expression vector, the one contains such nucleic acid sequence. Also includes the present invention one with such a vector transformed host cell and a method for production an essentially pure fusion protein for blocking the HIV Nef protein. Finally, the present invention relates therapeutic compositions comprising the foregoing Fusion proteins or nucleic acid sequences in combination with comprise a pharmaceutically acceptable carrier, and their Use in in vitro diagnosis / in vivo therapy of acquired immune deficiency syndrome (AIDS).

The Nef protein is a myristoylated "additional Protein "(accessory protein) of 27-35 kDa, which is only available in the Primate lentiviruses HIV-1, HIV-2 and SIV can be found. Early experiments on Rhesus macaques with SIV, the big one Deletions contained in the Nef gene showed that Nef for Maintaining optimal viral replication and for the course of the disease of AIDS is essential, and that a strong selection pressure functionally intact Nef variants in infected host (Kestler et al. 1991, Sawai et al. 1996, Khan et al., 1998). This role of Nef as one Key factor for lentiviral pathogenesis has been subsequently confirmed for HIV-1 in humans (Deacon et al., 1995, Kirchhoff et al. 1995).  

Although the importance of Nef for the viral life cycle became apparent through this work, its function at the molecular level is controversial (Cullen, 1998). A total of three functions have been assigned to the Nef protein:

• 1. changes the Nef protein through interaction with tyrosine and serine / threonine kinases the cellular signaling pathways (Baur et al. 1994, Iafrate et al. 1997, Hanna et al. 1998).
• 2. Nef increases viral infectivity at one stage the entry of the virus into the cell (Aiken and Trono, 1995; Schwartz et al. 1995).
• 3. Reduces the Nef protein by interacting with it Components of the endocytosis mechanism the expression of CD4 and antigens of the major histocompatibility complex Class I (Major Histocompatibility Complex Class I, MHC I) on the surface of infected cells (Garcia and Miller 1991; Aiken et al. 1994; Schwartz et al. 1996).

Given the high polymorphisms between different Nef alleles (Delassus et al. 1991) Preservation of these genetically separated functions important role of each individual function in the viral Life cycle close. How these functions are used Importance of the Nef protein in the infected host is contributing however largely unknown and remains the subject of others Research.

Proteolytic experiments showed that the Nef protein was a Has two-domain structure consisting of an N-terminal Membrane anchoring region and a well folded C- terminal core domain (Freund et al.  1994). Based on this observation, the structure determined by Nef for the nuclear domain by NMR spectroscopy (Grzesiek et al. 1996) and was also examined by X-ray Crystallography determined, each in complex with the SH3 Fyn tyrosine kinase domain (Lee et al., 1996; Arold et al., 1997) as well as alone (Arold et al., 1997). The Structures are in good agreement with each other, it is missing however, they have a flexible loop on the C- The term extends from remainder 159 to 173. Recently also became the N-terminal membrane anchoring domains structure in their myristoylated and non-myristoylated Form cleared, and showed a flexible polypeptide chain with two helical structural elements (Geyer et al., 1999). These two protein fragments now make it possible Gain insight into the full-length Nef protein and the proposed functions of the Nef protein with its reconciling structural features.

The HIV-1 / -2 and SIV-Nef protein is premature at one Infection of the cell expressed with HIV. 1995 was discovered that so-called long-term survivors who acquired under the Immune Deficiency Syndrome (AIDS) suffer and have been with for over 12 years HIV infected but do not develop an AIDS disease, have extensive deletions in HIV-Nef protein (Kirchhoff et al., 1995). The importance of the Nef protein for the HIV virus became clear.

Blocking the HIV-Nef protein is one promising path in the treatment of the acquired Immune deficiency syndrome. However, there currently exists in HIV Therapy is not a corresponding therapeutic approach against the HIV-Nef protein is targeted.

It is therefore an object of the present invention to be a protein provide that to block the HIV-Nef protein suitable is. This task is carried out in the Features specified independent claims solved. Further advantageous embodiments of the invention are in the dependent claims.

In the present invention, a Fusion protein or the coding for this protein To provide nucleic acid sequence that is highly specific the HIV-Nef protein binds and thereby blocks it.

The present invention is based on the observation that more in a structure-function comparison Sequences of the Nef protein from different HIV Patients with a typical course of the disease (i.e. none Long-term survivors) three sequential motifs occur that lie on the surface of the Nef protein and in all Sequences are highly conserved. First, these motives are one N-terminal myristoylation signal that the Membrane anchoring (and thus the localization) of the nep Proteins ensures, secondly, a PxxP protein sequence (P = Proline, x = any amino acid), the SH3 domains binds and thus typically a function in the Perceives signal transduction and thirdly a di-leucine Signal (LL), a motif that is typically the cellular Protein traffic controls traffic.

While the myristoylation signal is not very specific and occurs in a variety of eukaryotic proteins ( if this signal is attacked, a high frequency of undesirable effects would be expected) is the Combination of a PxxP binding motif and a di-leucine  Binding motifs (LL), such as those found in the HIV-Nef protein, unique and highly specific. In fact, the nef- Protein the only cytosolic and non-transmembrane Protein that is known to be a functional di- Owns leucine motif.

With the fusion protein according to the invention it has now been possible from this unique combination of two highly preserved Benefit and Amino Acid Sequence Motifs in Nef Protein to construct a highly specific ligand. This Ligand consists of a novel fusion protein, the both the LL binding domain and a PxxP Contains binding domain over a short Connection sequence (polypeptide linker) are connected. This Hybrid molecule has a high affinity to HIV Nef protein.

Since the Nef protein has no known human homolog (im Difference, for example to the HIV protease), is that here disclosed highly specific fusion protein, so that a effective therapeutic agent for the treatment of the acquired Immune deficiency syndrome (AIDS) in humans in comparison little or no adverse effects for the Is provided to patients. However, the invention is not limited to use in humans alone. The Fusion protein according to the invention can, for example, in each Mammal that is used with an HIV virus or is infected with an HIV-like virus (and that a nep- Homologous).

Basically, the fusion protein according to the invention is made up of the following components:

• a) a protein domain that has a di-leucine (LL) motif binds
• b) a protein domain that binds a PxxP motif, and
• c) a polypeptide linker through which the two Domains (a) and (b) are connected.

According to one embodiment of the invention, the Protein domain that binds a di-leucine motif through the Amino acid sequence from Seq. ID. No. 1 or homologue or Fragments thereof that have an LL binding activity maintained, defined.

According to a further embodiment, the PxxP motif binding protein domain through an SH3 domain with that in the Seq. ID. No. 2 defined amino acid sequence was formed. The Amino acid sequence of a complete fusion protein according to of the present invention is by Seq. ID. No. 3 Are defined.

Preferably, the fusion protein according to the present Invention additionally a signal for membrane anchoring contain. This signal is said to be specific and effective Increase fusion protein and target it to the membrane to which the HIV-Nef protein is also anchored.

According to a preferred embodiment, it is this signal around a C-terminal farnesylation signal, its sequence by Seq. ID. No. 4 is defined. Farther however, are also palmitoylation or myristoylation signals conceivable.  

According to a particularly preferred embodiment, this is Fusion protein according to the invention additionally with a signal equipped for degradation on the proteasome, where the Protein complex consisting of the fusion protein and HIV-Nef, is broken down. This signal preferably consists of a Ubiquitinylation signal, which by Seq. ID. No. 5 is defined.

The present invention further includes Fusion protein that contains the amino acid sequence of Seq. ID. No. 6 or homologs or fragments thereof, one maintain biological activity.

The present invention additionally relates to nucleic acid sequences for coding a fusion protein for blocking the HIV-Nef protein. These nucleic acid sequences basically consist of three components, namely

• 1. a nucleic acid sequence which is a di-leucine Encode (LL) motif binding protein domain,
• 2. a nucleic acid sequence that is a PxxP motif encode binding protein domain and
• 3. a nucleic acid sequence that contains a polypeptide linker encodes the two domains (a) and (b) are connected.

All or part of these nucleic acid sequences are in the Seq. ID. Nos. 7, 8, 9 and 10 defined.

For the specialist working in the relevant field obvious that the practice of the present invention  not limited to using the exact sequences is how they are in the Seq. ID. Nos. 1-10 are defined.

Modifications of the sequences, such as deletions, Insertions or substitutions in the sequence that are in the resulting protein molecule so-called "silent" changes (silent changes) are also considered within the Considered within the scope of the present invention.

As an example of this, changes in the Considered nucleic acid sequence that the generation of a equivalent amino acid at a given position Have consequence. Such amino acid Substitutions are the result of the replacement of an amino acid by a different amino acid with similar structural and / or chemical properties, d. H. conservative Amino acid replacements. Amino acid substitutions can be found on Basis of similarity in polarity, charge, Solubility, hydrophobicity, hydrophilicity, and / or the amphipathic (amphiphilic) nature of the residues involved be made. Examples of non-polar (hydrophobic) Amino acids are alanine, leucine, isoleucine, valine, proline, Phenylalanine, tryptophan and methionine. Polar neutral Amino acids include glycine, serine, threonine, cysteine, Thyrosine, asparagine and glutamine. Positively charged (Basic) amino acids include arginine, lysine and histidine on. And close negatively charged (acidic) amino acids Aspartic acid and glutamic acid.

"Insertions" or "deletions" typically move in the range of one to five amino acids. The allowed Degree of variation can be experimented through systematically insertions, deletions or substitutions made by  Amino acids in a polypeptide molecule using Recombinant DNA techniques and by examining oneself resulting recombinant variants with respect to their biological activity can be determined.

Nucleotide changes that are a change in the N-terminal and C-terminal portions of the protein molecule result, Often change protein activity because of these proportions usually not involved in biological activity are. It may also be desirable to use one or more of the to eliminate cysteines present in the sequence because the Presence of cysteines an undesirable formation of Multimers can result when the proteins are produced recombinantly, which makes cleaning and Crystallization process can be complicated. Each of the proposed modifications moves within the basic technical and scientific knowledge, just like determining the retention of biological Coded product activity.

It follows that where the term "DNA sequence" used either in the description or in the claims it includes all such modifications and variations, those in producing a biologically equivalent Fusion protein to block HIV-Nef result.

The present invention further relates to one Expression vector, one of the aforementioned Contains nucleic acid sequences.

Numerous vectors used for transforming of bacterial cells are known for example, plasmids and bacteriophages such as  for example the phage λ, as the most commonly used Vectors can be used for bacterial hosts. As well in For example, mammalian and insect cells can viral vectors for the expression of an exogenous DNA fragment be used. Exemplary vectors are the SV40 or Polyomavirus.

Alternatively, the host cells can be transformed directly by "naked DNA" without using a vector.

The fusion protein according to the invention can be produced either in eukaryotic cells or prokaryotic cells respectively. Examples of suitable eukaryotic cells include mammalian cells, plant cells, yeast cells and Insect cells. Appropriate prokaryotic hosts close E.coli and Bacillus subtilis.

The invention further relates to a method for production an essentially pure fusion protein for blocking the HIV-Nef protein that involves transforming a host cell a vector, cultivating the host cell under Conditions that require expression of the sequence by the Allow host cell and isolate the fusion protein from of the host cell.

Finally, the present invention comprises compositions which is an effective amount of a fusion protein as herein disclosed in combination with a pharmaceutical acceptable carrier include. Such a composition can also be by an effective amount of a nucleic acid sequence be educated.  

From the previously regarding the properties of the HIV nef- Protein and the function of the invention Fusion protein stated above shows that this Compositions for in vivo therapy of the acquired Immune deficiency syndrome (AIDS) are suitable. However, it is also possible to use these compositions for in vitro diagnosis of acquired acquired immunodeficiency syndrome. In the latter case is a connection of the fusion proteins / Nucleic acid sequences with a suitable detection reagent advantageous in that the presence of the HIV Nef protein in can be demonstrated using a test kit.

The present invention is based on the enclosed pictures described. The pictures show:

Fig. 1 shows the structure-function relationship of the highly conserved motifs for myristoylation (MGxxxS), SH3-domain binding (PxxP) and LL-binding domain binding (LL) in HIV-Nef.

Fig. 2 is a schematic representation of the modular domain arrangement in a fusion protein for blocking the HIV Nef protein.

Fig. 3 that the fusion protein consisting of an LL domain and an SH3 domain binds specifically to the HIV Nef protein.

In Fig. 1, A) shows the three-dimensional protein structure of HIV-1 Nef with the labeled highly conserved motifs for protein interaction, which illustrates the exposure of the binding sites. The sequence motif MGxxxS at the N-terminus of Nef initiates the co-translational myristoylation of the protein and thus membrane independence. The polyproline helix (PxxP motif or as an exact description: PxxPxR) leads to the recognition of SH3 domains while the structurally opposite di-leucine motif (LL motif or: ExxxLL) interacts with the di-leucine binding domain. The representation of the full-length protein structure of Nef is based on the composition of the structures of the anchor domain (amino acids 1-57; Geyer et al., 1999) and the core domain (amino acids 57-206; Grzesiek et al., 1996), which by means of Nuclear magnetic resonance (NMR) spectroscopy have been determined and energy-minimized with subsequent molecular dynamics.

B) shows the correlation of sequence conservation and Surface accessibility that the structural Functional relationship of the HIV Nef protein clarified. Amino acids that play a key role in folding The protein structure is above average preserved and hidden inside the structure (see e.g. Trp-183). In contrast, amino acids are the are heavily exposed but are still highly preserved, often important for protein interactions (see LL- Motif, 164 165). The for the functionality of the viral Protein's important motives can be identified in this way.

The degree of sequence conservation was from an alignment of 186 individual Nef amino acid sequences different HIV-1 subtypes determined. The surface accessibility of the individual amino acids, divided into main and side chain, was based on the Nef protein structure shown in A) calculated.  

In FIG. 2, A) shows the LL-binding domain (LL domain), which comprises approximately 350 amino acids, and which via a polypeptide linker of variable length (10 to 60 amino acids) with a 61 amino acid PxxP-binding domain (SH3 domain) is connected. The specificity of this hybrid molecule for blocking the HIV Nef protein results from the chimera created here of a domain from vesicular transport processes (LL domain) with a domain from signal transduction processes (SH3 domain).

The resulting fusion protein specifically targets the two motifs of the highest conservation (LL and PxxP) in Nef, that come from a very different cellular context.

B) of Fig. 2 shows a sketch of a fusion protein with the reverse arrangement of the protein domains. C) The fusion protein for blocking the HIV Nef protein can be further specified and optimized by introducing two additional sequential motifs. A farnesylation motif at the C-terminus for membrane anchoring (or palmitoylation motif, myristoylation motif) ensures the localization of the fusion protein in the vicinity of the membrane-bound Nef protein. A signal for ubiquitinylation (or also sumoylation) can direct the complex from the fusion protein with Nef to break down at the proteasome of the cell. The arrangement of the domains, linkers and additional motifs must be optimized for the greatest affinity, specificity and functionality for blocking the HIV Nef protein.

Binding experiments ( Fig. 3)

Two different Nef proteins from the two most commonly used HIV-1 strains NL4-3 and SF2 in the laboratory were used for the binding experiments. The two Nef proteins were expressed as fusion proteins with glutathione-S-transferase (GST) in Echerichia Coli and purified using GST-Sepharose beads. The fusion protein consisting of the LL domain of the β-subunit of the adapter-protein complex AP-1 (β-1) and the PxxP-binding SH3 domain of the tyrosine kinase Hck (SH3-Hck) was made with two different lengths of the Linker cloned (see Fig. 2). The use of the two linkers of 60 amino acids in length and 106 amino acids in length leads to the two variants of the fusion protein LL-SH3 (470) and LL-SH3 (516) with a running behavior of 48 kDa and 56 kDa, respectively. [In detail, the chimera consists of AC: AAA40807 (β1), AS 171-578 (LL domain and linker) or AS 171-624, -Threonin- (for the restriction interface) and AC: P08631, AS 78-138 ( Hck-SH3).] The fusion proteins were transcribed in vitro and translated with the TnT T7 coupled reticulocyte lysate system (Promega, Wisconsin) in the presence of [ ³⁵ S] -labeled cysteine.

The binding assay between GST-Nef and in vitro translated fusion protein was in standard kinase Extraction buffer (KEB) performed (250 mM NaCL, 0.5% NP40, 2 mM EDTA, 10% glycerol, 50 mM Tris-HCl (pH 7.4) and 1 mg / ml Proteinase inhibitor cocktail). About 4 µl of the GST-Nef Protein was mixed with 7 µl of the fusion protein over 3 hours Shaken slowly at 4 ° C in 500 µl KEB. Subsequently the GST beads were washed three times in KEB, the proteins  separated by 10% SDS-PAGE gel electrophoresis and by Autoradiography made visible.

The binding experiment shows that the two fusion proteins different lengths specific to the HIV-1 Nef protein of alleles NL4-3 and SF2 bind (lanes 2, 3 and 5, 6) during no binding between GST alone and the fusion proteins can be detected (lane 1, 4). The high affinity of the Binding is represented by 10% of the input of the Fusion proteins clearly (lanes 7, 8).

literature

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SEQUENCE LISTING

Claims (24)

1. Fusion protein for blocking the HIV-Nef protein, which consists of the following:

• a) a protein domain that binds a di-leucine (LL) motif,
• b) a protein domain that binds a PxxP motif, and
• c) a polypeptide linker through which the two domains (a) and (b) are connected.

2. Fusion protein according to claim 1, wherein the protein domain of a) the amino acid sequence of Seq. ID. No. 1 or homologue or fragments thereof which have an LL binding activity maintained. 3. Fusion protein according to claim 1 or 2, wherein the Protein domain of b) an SH3 domain with the amino acid sequence by Seq. ID. No. 2 or homologues or fragments thereof which maintain PxxP binding activity. 4. Fusion protein that contains the amino acid sequence of Seq. ID. No. 3 or homologs or fragments thereof, one maintain biological activity. 5. fusion protein according to any one of the preceding claims, which also contains a signal for membrane anchoring. 6. Fusion protein according to claim 5, wherein the signal is a C- terminal farnesylation signal according to Seq. ID. No. 4 is. 7. Fusion protein according to one of the preceding claims, which is also a signal for degradation on the proteasome contains.   8. Fusion protein according to claim 7, wherein the signal Ubiquitinylation signal according to Seq. ID. No. 5 is. 9. Fusion protein that contains the amino acid sequence of Seq. ID. No. 6 or homologs or fragments thereof, one maintain biological activity. 10. Nucleic acid sequence for coding a fusion protein for blocking the HIV-Nef protein, which consists of the following in a functional connection:

• a) a nucleic acid sequence encoding a protein domain that binds a di-leucine (LL) motif,
• b) a nucleic acid sequence encoding a protein domain that binds a PxxP motif, and
• c) a nucleic acid sequence encoding a polypeptide linker through which the two. Domains (a) and (b) are connected.

11. The nucleic acid sequence according to claim 10, wherein the Nucleic acid sequence from a) the sequence from Seq. ID. No. 7 or includes a portion thereof that contains a protein domain with LL Encoding binding activity. 12. Nucleic acid sequence according to claim 10 or 11, wherein the Nucleic acid sequence from b) the sequence from Seq. ID. No. 8 or includes a portion thereof that contains a protein domain with PxxP Encoding binding activity. 13. Nucleic acid sequence according to claim 10, which the Seq. ID. No. 9 or a part thereof, which is a biologically active Fusion protein encoded. 14. Nucleic acid sequence according to any one of claims 10-13, the additionally encoded a signal for membrane anchoring.   15. nucleic acid sequence according to claim 10-14, the additional encodes a degradation signal on the proteasome. 16. Nucleic acid sequence that the Seq. ID. No. 10 or one Part of which includes a biologically active Fusion protein encoded. 17. Nucleic acid sequence that is the complementary sequence of one of the Nucleic acid sequences of claims 10-16. 18. Expression vector of a nucleic acid sequence Claim 10-17 contains. 19. A host cell containing a vector according to claim 18 is transformed. 20. Process for producing a substantially pure Fusion protein for blocking the HIV Nef protein, which is a Transforming a host cell with a vector according to claim 18, cultivating the host cell under conditions that a Allow expression of the sequence by the host cell, and a Isolate the fusion protein from the host cell. 21. Therapeutic composition that is an effective amount of a fusion protein according to any one of claims 1-9, in combination with a pharmaceutically acceptable carrier. 22. Therapeutic composition that is an effective amount a nucleic acid sequence according to claims 10-17, in Combination with a pharmaceutically acceptable carrier. 23. Use of the compositions according to claims 21 and 22 in the in vitro diagnosis of acquired immune deficiency syndrome (AIDS). 24. Use of the compositions according to claims 21 and 22 for in vivo therapy of acquired immune deficiency syndrome (AIDS) in humans.