JP2001518295A - Methods for selectively controlling membrane protein display and protein secretion in eukaryotic cells - Google Patents

Abstract

  (57) [Summary] Modulate the presence of selected essential membrane proteins on the cell surface membrane by increasing or decreasing the intracellular transport of proteins from the endoplasmic reticulum to and through the cis-Golgi apparatus; or Disclosed are methods of modulating secretion of selected secreted proteins and related compositions. Disclosed are related methods for determining whether the intracellular transport of a particular protein is mediated by the spectrin-ankyrin-adaptor protein transport system (SAATS). Also disclosed are related methods of determining whether a candidate compound inhibits or enhances the intracellular transport of a selected protein from the endoplasmic reticulum to the cis-Golgi apparatus by SAATS. The disclosed agents and methods are applicable to a variety of uses, including immunoregulators, ion transport inhibitors, vascular modulators and cancer chemotherapeutics.

Description

DETAILED DESCRIPTION OF THE INVENTION

Related Application This application is hereby incorporated by reference in its entirety, May 2, 1997.
It relates to provisional application 60 / 047,352 filed on 1 day and provisional application 60 / 053,218 filed on July 18, 1997. This application also claims priority to US Provisional Application No. 60 / 060,559, which is incorporated herein by reference in its entirety. All other articles, publications, patents and references cited throughout this application are hereby incorporated by reference in their entirety.

[0002]

TECHNICAL FIELD OF THE INVENTION

The present application describes the sequestration of essential membrane or secreted proteins into transport vesicles and selectively modulates the transport of such vesicles between such endoplasmic reticulum, Golgi apparatus, plasma membrane and other membrane compartments About the method. The present invention, based on the discovery of new biological phenomena, inhibits the methods and compositions used to identify important essential membrane and secretory proteins, and the sequestration of such proteins into transport vesicles Provide the drug. Related methods and compositions can be used to modulate secretion and cell membrane display of various disease-related proteins.

[0003] Acknowledgments of Federal Government Support [0003] The research and findings described herein are provided by the National Institutes of
Health: NIH P01-DK38979, NIH R01-HL28560,
Supported by grants from NIH R01-DK43812 and NIH R29-DK47072.

[0004]

[Prior art]

In all eukaryotic cells, a central and common process in the delivery of essential membrane and secretory proteins is from the endoplasmic reticulum to the Golgi apparatus, and finally through the site of their synthesis, and finally Is vesicle transport to specific plasma membrane or inner membrane domains. For example, Molecular Cell
Biology (1990, Scientific American
Books), Chapter 17, Plasma-Membrane, Secreto
ry, and Lysosome Proteins: Biosynthe
See sis and Sorting. The general mechanism by which this process is accomplished is now understood in considerable detail and involves a complex pathway of coated vesicle budding, transport, and fusion (Kreis and Peppe).
rkok, 1994; Schmid and Damke, 1995;
Rothman and Wieland, 1996; Skekman
and Orci, 1996; Bannyth and Balch, 1
997). Collectively, these processes coordinate the synthesis and folding of membrane proteins in the endoplasmic reticulum, sequential vesicle trafficking through various Golgi compartments, and finally their targeting to specific plasma membranes or organelle domains. Linked to the process of chemical delivery. The molecular mechanisms that mediate these processes are not well understood. Typically, budding vesicles are encapsulated in a fairly adherent protein shell that favors their extrusion from planar membrane compartments. Three general types of coats are recognized today. That is, clatrin / AP, COPI, and COPII. These coats are similar in many ways. These all form an easily identifiable electron density layer around each of these vesicles; they are relatively simple and uniform in composition, and they are ARF-like GTP-
Assembles on vesicles under the control of the binding protein.

[0005] Homologs of the erythrocyte spectin-ankyrin membrane cytoskeleton have recently emerged as candidates for a fourth class of vesicle or Golgi coat proteins. Immunological, gel electrophoresis and functional data strongly suggest the presence of a novel isoform of spectin associated with the Golgi membrane, defined as βIΣ ^* and βIII (Beck et al., 1994; for review, Devarajan And Morrow,
1996; Devarajan et al., 1996; Morrow et al., 199.
7). Such isoforms of spectin have been identified as components of the dynactin complex required for microtubule-based vesicle trafficking (Holleran et al.,
1996). Adapter proteins that link the unique analogs of ankyrin, Na, K-ATPae and other transport proteins, to the spectin backbone of the plasma membrane (
For a review, see Bennett, 1992; Devarajan and M.
orrow, 1996; see Morrwo et al., 1997) also associated with the Golgi. The Golgi-associated ankyrin is a novel small ankyrin that has been cloned and characterized (Ank _G119 ) (Devarajan et al., 19).
96), and the larger isoform (B
Eck et al., 1997). Additional ankyrins also associate with other inner membrane compartments such as lysosomes (Hookock et al., 1997). Spectin differs from coatomer protein in that it does not form a geometrically accurate coat and is more difficult to visualize by electron microscopy, but, like other coat proteins, interacts with the Golgi membrane. meeting Supekuchin is, PtdInsP ₂ - Thus is stimulated dependent manner ARF (Godi et al., 1997). The evidence disclosed below:
Figure 2 shows a direct and specific role for spectin-ankyrin and other adapter proteins in mediating the transport of endogenous and secreted proteins.
This Supekuchin - ankyrin - adapter transport (s pectin- a nkyri
n- a dapter p rotein t raffucking) / coupling system,
Collectively called SAATS.

[0006]

[Means for Solving the Problems]

The present invention relates to transport from the endoplasmic reticulum to the cis-Golgi apparatus, or from the cis-Golgi to the medial-Golgi apparatus, or from the medial-Golgi apparatus to the trans-Golgi apparatus, or from the trans-Golgi apparatus to the cell membrane or other cellular compartment. Based on the discovery of covert intracellular processing and transport systems that mediate the sequestration of essential membrane or secretory proteins into transport vesicles. We have discovered that upon isolation, adapter proteins and functionally active sites within pharmacologically active components of the SAATS system with the ability to specifically control the display or transport of selected membrane proteins. Is identified. A general method is provided that allows specific transport inhibitors for selected membrane or secreted proteins to be identified, and some examples of the application of these methods are provided.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION

The following discussion provides a general description of the SAATS system, which can modulate the general class of essential and secretory proteins, as well as specific proteins to affect cell membrane display and secretion. Consider the method.

The generality of which class of membrane proteins utilizes this pathway remains to be well determined. However, given the guidance provided herein, one of ordinary skill in the art would know how to determine which proteins participate in the intracellular transport system of SAATS and homologues, the ER to and through the Golgi apparatus. Methods for identifying vesicle skeletal proteins and associated adapter proteins that mediate protein transport from proteins, methods for identifying small molecules and other agents that mediate such transport, and methods of using such agents for therapeutic purposes. You will know.

Table IA: Summary of Mammalian Spectrin

Table IB: Interactions involving spectin

1. Bennet and Branton, 1977; Bennet and Stenbuck, 1979b; Bennet and Ste
nbuck, 1979a; Morgans and Kopito, 1993. 2. Nelson and Veshnock, 1987; Koob et al., 1988; Morrow et al., 1989; Nelson and Hammerton, 1989. 3. Smith et al., 1991. 4. Sr.
inivasan et al., 1988; Srinivasan et al., 1992. 5. Luna and Hitt, 1992. 6. Joseph and Samanta, 1993. 7. Lokeshwar and Bourguignon, 1992a; Lockeshwar et al., 1994.
8. Bourguignon et al., 1992. 9. Davis and Bennet, 1993; Davis et al., 1993. 10. Sm
ith et al., 1993. 11. Hemming et al., 1995. 12. Bourguignon et al., 1986. 13. Lombardo et al., 1
994a. 14. Sinard et al., 1994. 15. Pitcher et al., 1992; Touhara et al., 1994. 16. Bourgui
gnon et al., 1985; Lokeshwar and Bourguignon, 1992b. 17.Molday et al., 1990. 18. Ci
anci et al., 1995. 19. Rotin, et al. 1994. 20. Pollerberg et al., 1987. 21. Mombers et al., 1977
; Mombers et al., 1979; Pradhan et al., 1991; Diakowski and Sikorski, 1994. 22. Andr
ews and Fox, 1992; Lopez et al., 1992. 23. Rimm et al., 1995. 24. Gardner and Benne
tt, 1987; Mische et al., 1987. 25. Cohen and Foley, 1980; Cohen and Korsgren, 1
980; Cohen and Foley, 1982; Wolfe et al., 1982. 26. Mangeat and Burridge, 1984.
Langley and Cohen; Frappier et al., 1987; Herrmann and Wiche, 1987; Frappie.
Fach et al., 1985; Riederer and Goodman, 1990. 28.Herrmann and Wiche, 1987. 29. Sikorski et al., 1991. 30. Hayes et al., 1995. 31. Bahler and Gre.
engard, 1987; Petrucci and Morrow, 1987. 32. Fowler and Bennet, 1984; Fow
ler et al., 1993. 33. Siegel and Branton, 1985; Husain-Chishiti et al., 1988. 34. Ma.
rfatia et al., 1994; Hemming et al., 1995; Marfatia et al., 1995.35.Georgatos and March
esi, 1985: Georgatos et al., 1985. 36. Hyvonen et al., 1995; Wang and Shaw, 1995.

[0012] The practice of the present invention thus generally involves the modulation of specific interactions between SAATS and ER and Golgi recognized cargo or transport proteins.
Also described is the elucidation of specific sites (often complex and containing homologs of spectrin + adapter proteins) to identify targets for blocking specific interactions. In some cases, the SAATS system is H, K-ATPase, N
a, It is possible to distinguish between K-ATPase, CFTR and other transport molecules, or to modulate at the level of a binding domain common to various classes of proteins.

[0013] For example, identifying the binding domain for CFTR (gene product responsible for cystic fibrosis) in SAATS by the techniques disclosed herein has been demonstrated in the plasma membrane of mutated (but functional) CFTR. Enhanced delivery to the drug will allow for an initial analysis of possible transport-enhancing agents that may reduce the clinical severity of the disease. In Figures 12 and 22, binding of the CFTR to the SAATS ankyrin component and its enhanced delivery to the membrane by the βIN _-5,15 construct is shown.

[0014] An important finding under investigation is the actual genetic makeup of spectrin (s) involved in SAATS. While not intending to be bound by the identification of any specific spectrin species, we believe that homologues of βI spectrin, such as βI spectrin or βIII spectrin, are involved in the SAATS process. Because they have a broad immunological and functional similarity to SAATS spectrin, peptides based on βI spectrin (Win
Kelmann et al., 1990) are functionally active as discussed below. A discussion of the specific Golgi-association spectrum, referred to as βIΣ ^* , is still found in our earlier publications, because it is still a somewhat uncharacterized putative splice form of βI spectrin. That is, Devarajan et al., JCB 1
33: 819-830 (1996) and Beck et al. (1994).

Various adapter proteins other than ankyrin, such as protein 4.1 homolog, adsin homolog, catenin homolog, and the like may be involved. One of skill in the art will elucidate the role of such adapter proteins by the techniques disclosed herein to determine their involvement in modulating the interaction of SAATS with ER and proteins carried through the Golgi. be able to. It is likely that similar pathways that affect the delivery of proteins from the medial-Golgi and trans-Golgi to the plasma membrane or other inner membrane-associated compartment are likely, but will not be sufficient for the techniques disclosed herein. Wait for elucidation.

The methods outlined here provide a novel approach to control the activity of a specific membrane protein if it is a receptor, adhesion receptor, ion channel, or transporter. While most conventional therapeutic approaches target the activity of a given protein, the approaches outlined here target the exact appearance of the protein at the cell surface or at secretory vesicles. This strategy is somewhat similar in concept to approaches that attempt to specifically repress the synthesis of a given protein, such as by antisense RNA or by a specific transcriptional regulator. However, the methods outlined here are essentially that they do not attempt to 1) inhibit the synthesis of a given protein, but only its delivery to the correct cell or tissue compartment, 2) RNA 3) It is fundamentally different in that it does not require intracellular expression and reduces the inherent obstacles in such a task. 3) It should be suitable for high-throughput in vitro screening assay and suitable for regulation by small molecule effector. In this way, the methods outlined herein complement and represent a novel therapeutic approach that is synergistic with other representative drug development strategies.

As used herein, the following terms have defined meanings.

A. As used herein, a transport vesicle refers to any small membrane-associated structure that contains a protein and is involved in the transfer of such protein from one membrane compartment to another.

B. As used herein, cell membrane refers to lipids and proteins that contain a bilayer structure that binds to any cell compartment, including the cell surface, the so-called plasma membrane.

C. As used herein, an adapter protein is defined as direct to the SAATS spectin scaffold or to the transport protein or any other multifunctional protein that directly or indirectly determines the specificity of capture of the transport protein carrier by SAATS. Or any protein that provides indirect binding.

D. As used herein, randomly selected refers to the case where the agent is randomly selected without considering the specific sequences of the vesicle skeletal protein and the associated adapter protein. Examples of randomly selected agents are the use of chemical or peptide combinatorial libraries, or growth broths of biological or plant extracts.

E. As used herein, rationally selected or rationally designed is selected on a non-random basis taking into account the sequence of the target site and / or the conformation associated with the action of the drug. Point. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the relevant vesicle backbone protein or associated adapter protein. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to a fragment or related domain of a spectrin protein.

F. As used herein, without further designation, "cell" means any eukaryotic cell population, including primary and transformed immortalized cell lines that express spectrin or an equivalent protein. Madin Darby canine kidney (MDCK) cells can be used in the claimed method. Transfection of some cell lines with broadly active SAATS inhibitors, such as βIN _-5 or βIN _-2 constructs, can lead to poor cell survival and activation of apoptosis or lethal swelling. The problem is often that SAATS agents with less extensive activity (eg, larger constructs or only spectrin, a small portion of ankyrin were deleted, or one of the adapter proteins was deleted or modified) Construct). This strategy of scanning mutagenesis will minimize global disrupted cell trafficking with the worst disruptors of SAATS and improve cell viability so that measurements can be made on less robust cell lines. This approach also has the advantage of more accurately locating the SAATS activity for a given protein. Transfection levels can be modified or controlled to manage poor cell survival.

[0024] If induction of apoptosis is a problem with some widely active SAATS constructs, improvements in the viability of cell-based assays may require the use of available apoptosis inhibitors such as caspase inhibitors or Fas blocking antibodies. (US Pat. Nos. 5,632,994, 5,656,725, 5650,491 and 5,635,187).
No.). In addition, cells can be designed to express increased levels of bcl-2 (see US Patent No. 5,650,491).

G. As used herein, “stringent conditions” refers to Sambrook et al. (Molecular Cloning: A Laboratory Appr.)
oach, Cold Spring Harbor Press, NY,
1989) or Ausubel et al. (Current Protocols)
in Molecular Biology, Green Publish
ing Co. , NY, 1995) refers to conditions that are normally defined and available.

Hybridization, apart from other variables, is based on sequence identity (homology), G + C content of the sequence, buffer salt content, sequence length and duplex melting temperature (T
[M]). For example, Maniatis et al. (Molecular C
loning: Cold Spring Habor Laboratory,
Cold Spring Harbor, NY, 1982). Given similar sequence lengths, buffer salt concentration and temperature provide useful variables for assessing sequence identity (homology) by hybridization techniques. For example, if there is at least 90 percent homology, hybridization is usually performed at 68 ° C. in a buffer such as 6 × SCC diluted from 20 × SSC. See Maniatis et al. The buffer salts utilized in the final Southern blot wash can be used at low concentrations, eg, 0.1 × SSC, at relatively high temperatures, eg, 68 ° C., and the two sequences form a hybrid duplex (Hybridize). The combined use of said hybridization and washing conditions is defined as high stringency conditions or high stringency conditions. Moderately high stringency conditions can be used in hybridizations where the two sequences have at least about 80 percent homology. Here, hybridization is performed using 6 × SSC at a temperature of about 50 to 55 ° C. A final wash salt concentration of about 1-3XSSC and a temperature of about 60-68C are used. These hybridization and wash conditions define moderately high stringency conditions.

In particular, specific hybridization refers to conditions in which a high degree of complementarity exists between a nucleic acid consisting of at least one sequence of the figures and another nucleic acid.
In selective hybridization, complementarity is generally at least about 75%,
80%, 85%, preferably about 90 to 100%, most preferably about 95 to 1
00%.

I. General Description The present invention relates to the transport of essential and secretory proteins by controlling their sequestration into transport vesicles that mediate protein transport between the ER, Golgi, plasma membrane and other membrane compartments And a method for selectively controlling. The present patent application extends the discovery that, by multiple interactions, specific isoforms of spectrin and various adapter proteins, such as ankyrin, associate with vesicles and the Golgi membrane (Devarajan et al., 1994; Devarajan and Morrow, 1995; Devarajan and Morrow, 19
96; Devarajan et al., 1996a; Devarajan et al., 1
996b; Devarajan et al., 1997; Godi et al., 1997).
These interactions are required for the transport of a broad class (and possibly all) of membrane and secreted proteins from the ER to the medial-Golgi via the intermediate compartment (
Devarajan et al., 1996a; Devarajan et al., 1997;
Godi et al., 1997). In addition, by modifying specific interactions within spectrin or in one of the associated adapter proteins, significantly alter or block the transport and ultimately the rate of delivery of the specific protein to its final membrane target. can do.

We refer to this newly discovered system that mediates transport as the spectrin-ankyrin-adaptor protein transport system (SAATS). All membrane and secretory proteins must be selectively and sequentially collected in different transport vesicles during their transfer from the ER to the plasma membrane (or to other membrane compartments) via this pathway As such, this system has wide potential applicability and generality. We believe that the large diversity of ER-derived proteins is due to a combined process involving many weak but specific interactions, created by the multivalent binding capacity of spectrin and ankyrin and other adapter proteins. , As well as by pairing of potentially unbound and binding proteins in the oligomer complex. We believe that the regulation of secretory protein trafficking through the recognized cargo loading proteins present in the ER is mediated by this pathway, where the cargo loading membrane proteins are spectrin / Binds to the adapter protein system.

Thus, (but not other proteins) the plasma membrane (
Or other targeted membrane compartments) by selectively controlling the binding of specific membrane proteins, or the binding of a particular set of cargo proteins to SAATS. Delivery of the protein to the transport vesicles will be modified (blocked or enhanced). The SAATS system and its functions are shown in FIG.

We also believe that a similar but distinct SAATS system is involved in protein transport between the ER and the Golgi, also from the Golgi to the plasma membrane or other target (ie, post-Golgi). Think of it as mediating. Thus, the methods described herein apply equally to targeted membrane delivery after the Golgi. An example of such a process would be spectrin-linked microtubule-mediated axial transport of specific neuronal receptors that are destined for the pre- or postsynaptic membrane.

In general, a number of methods are known for binding bioproteins (or lipids) to SAATS using methods and techniques known in the biomedical arts but conventionally applied for other purposes. Available to control.

A) Recombinant cDNA methods, such as maintaining the actin binding motif of MAD1 and βI spectrin (Lombardo et al., 1994), but (Na, K
Deletion of its ankyrin binding domain (binding to ATPase) (Kenneth
dy et al., 1991), selectively blocks the delivery of Na, K-ATPase to the plasma membrane (Devarajan et al., 1996a; Devarajan et al., 1991).
1997) Use of expression of βI spectrin-related peptides and homologs from other spectrin-related genes. Since other proteins are involved in the SAATS transport process, this may result in sequestration of selected membranes and secreted proteins into transport vesicles and ultimately their display on the plasma membrane, or through the plasma membrane This opens up the possibility of a gene therapy approach to modulation of SAATS function to introduce a gene encoding a modified spectrin that either inhibits or enhances the secretion of SATS.

B) Certain polyclonal and monoclonal antibodies to spectrin block transport of Na, K-ATPase (FIG. 18) or VSV-G protein (Harris et al., 1986; Devarajan et al., 1996a)
Godi et al., 1997). Although such drugs generally have extra-cellular effects and therefore have limited therapeutic goals, the present inventors have identified the importance of such drugs in in vitro transport assays and immunomicroscopy. Think of it as a useful tool.

C) Inhibitors of ADP-ribosylation factor (ARF) block the binding of the PH domain of βI spectrin through a PtdInsP ₂ -dependent process, which allows SAATS to organize specific phospholipids and It also means that it contributes to transportation.
Agents that directly modulate PtdInsP ₂ phospholipid display, or ARF activity, can thus form the basis for a class of therapeutic agents. Examples of such agents include GTs such as permeant GTP-γ-S
It may be a permeable non-hydrolysable homolog of P.

D) We also block the transport of selected essential membrane and secretory proteins (FIG., Godi et al., 1997) Specific functional domains of βI spectrin (Kennedy et al., 1991; Lombardo et al.) , 1994
Weed et al., 1996) were prepared. These demonstrate the potential activity of small peptide or peptidomimetic drugs.

E) Based on our understanding of the spectrin scaffold present at the plasma membrane (for a review see Morrow et al., 1997), many of the interactions involving SAATS involve protein kinase C , Ca ++, calmodulin, casein kinase, etc., appear to be subject to post-translational regulation. Therefore,
Agents that modulate these regulatory pathways, although less specific, can form the basis for potential SAATS-directed therapeutic agents.

F) We believe that small molecule modulators of SAATS-specific interactions will form the basis for a broad and effective category of therapeutic agents. Such small molecule agents will either block or enhance the delivery of specific proteins to the plasma membrane or other site. We have developed several in vitro assays to detect specific interactions between SAATS and other proteins based on in vitro binding, surface plasmon resonance, and genetic screening (2-hybrid system). Was developed. Many of these in vitro assays are
Facilitates lead compound identification according to high-throughput screening. We have also developed advanced algorithms that allow the creation of three-dimensional structures of certain spectrin repeat units along with mutant sequences that mediate specific binding interactions (Cianci). And Morrow, 1997; Gallag.
Her et al., 1997; Stabach et al., 1997). We believe that the availability of these advanced 3-D protein models will facilitate rational drug design initiatives.

The specific examples presented below are illustrative only and are not intended to limit the scope of the present invention.

II. Specific Embodiments The following discussion describes certain embodiments of our invention, in which (A) its vesicle transport to or through the Golgi is mediated by SAATS or similar systems (B) identifying any essential vesicle skeletal proteins, ie, spectrin or associated adapter proteins, ie, ankyrins, into the transport vesicles of the specific essential membrane and secretory proteins; Identify peptides and small molecules that modulate the sequestration and / or vesicle trafficking of such proteins; and (D) identify such peptides for therapeutic and research purposes. Procedures are provided for utilizing agents that modulate protein sequestration and / or vesicle trafficking.

[0041]

【Example】

Example 1: Identification of essential membrane and secretory proteins whose vesicle trafficking is mediated by SAATS The identification of broad inhibitors of SAATS generally involves the cultivation of certain specific essential membrane or secretory proteins. One can approach by comparing the ability of a series of overlapping and complementary spectrin peptides to disrupt transport in cell lines. This information allows the identification of the smallest region within spectrin that is responsible for the potential sorting and docking of targeted proteins into the SAATS system. Other potential blocking agents also follow from the knowledge of adapter proteins that bind to this region of spectrin, as described below. The following steps are preferred.

Step A1: A model cell line expressing the membrane or secreted protein that is to be evaluated and that allows the detection of surface or secreted target proteins exists for which the membrane or secreted protein being evaluated is present Select Examples are immunofluorescent detection of surface proteins; flow cytometry; surface labeling assay; or pulsed ³⁵ S
This would be a transport assay using a methionine label. Assays to monitor the transport of specific proteins can also be performed by using GFP-labeled proteins and time-lapse biofluorescence microscopy. Also, detection of specific transport or electrical activity associated with surface display or secretion of the targeted protein is contemplated. As a specific example, cultured Madin Darby canine kidney cells (MDCK) can be used to monitor surface expression of Na, K-ATPase by indirect immunofluorescence (see FIGS. 3-10, 18).

Step A2: Individual colony lines transfected with retroviral vectors or other virus-based infection strategies or transiently transfected using standard transfection methods of model cell lines Prepare the system,
Each expresses one of a complementary and overlapping series of βIΣ ^* spectrin cDNA constructs of various lengths under the control of a powerful eukaryotic expression vector. A series of suitable βIΣ ^* spectrin constructs are listed in FIG. For example, one suitable system is a stable MDCK cell line that expresses a βIN _-5 spectrin peptide (see FIG. 3, etc.) that competitively replaces or replaces the endogenous SAATS system.

Transformation of a suitable cellular host with an rDNA (recombinant DNA) molecule of the present invention is typically accomplished by well-known methods, depending on the type of vector used and the host system used. Is done. For transformation of prokaryotic host cells, electroporation and salt treatment methods are typically used. For example, Cohe
n et al., Proc. Acad. Sci. USA (1972) 69:21.
10; and Maniatis et al., Molecular Cloning,
A Laboratory Manual, Cold Spring Har
Bor Laboratory, Cold Spring Harbor,
NY (1982). For methods of transforming vertebrate cells with vectors containing rDNA, electroporation, cationic lipid or salt treatment methods are typically used. See, for example, Graham et al., Virol (19
73) 52: 456; Wigler et al., Proc. Natl. Acad
. Sci. USA (1979) 76: 1373-76.

[0045] Successfully transformed cells, ie, cells that contain a rDNA molecule of the present invention, can be identified by well-known techniques. For example, cells obtained from the introduction of the rDNA of the present invention can be cloned to obtain a single colony. Cells from those colonies are harvested, lysed, and their DNA content determined, for example, by Southern, J. et al. Mol. Biol. (1975) 98:50
3, or examined for the presence of rDNA using the method described by Bernett et al., Biotech (1985) 3: 208, or assay the protein produced from cells through immunological methods. If, for example, a tag such as green fluorescent protein is used in the construction of the recombinant DNA, the transfected cells can also be detected in vivo by the fluorescence of such molecules by cell sorting. .

Step A3: Evaluate each clonal line or cell expressing the desired construct for disruption of surface display or secretion of the selected protein (assays where transient expression is used, eg, FIG. 9). Expression of full-length βIΣ ^* spectrin (or other full-length spectrin) will not disrupt SAATS. Expression of at least one of the truncated constructs shown in FIG. 2 or similar constructs for other spectrins and other vesicle skeletal proteins will act in a dominant negative manner to disrupt SAATS. . Comparison of the inhibitory activity of different constructs will reveal the boundaries of inhibitory peptide or cDNA constructs encoding such inhibitory peptides. For example, inhibition of surface display of Na, K-ATPase in MDCK cells by expression of βIN _-5 spectrin construct (FIG. 3).

Step A4: Knowledge of minimal inhibitory peptides was further prepared from βIΣ ^* spectrin constructs or other spectrins in which specific regions deleted in the inhibitory construct reverted to the inhibitory construct. Further refinement can be achieved by repeating steps A2 and A3 above with similar constructs. Thereby selected (targeted)
Identify the regions required to rescue the surface display or secretion of the protein. This approach allows the identification of the minimum sequence required to dock a protein targeted to SAATS.

For example, surface display of Na, K-ATPase in MDCK cells, which is inhibited by βI _N-5 , is rescued by attaching βI spectrin repeats 14 and 15 to the βI _{N- 5} construct. (See FIG. 6 and others). These results indicate that βI spectrin, the 14-15 repeat region, has Na,
This indicates that it is a docking site (directly or indirectly) of K-ATPase.

Step A5: A more detailed analysis of the SAATS docking site for the targeted protein, whether it directly attaches spectrin at the site identified in steps A1-A4, or utilizes an intermediate adapter protein It can be obtained by deciding whether or not. In most cases, this can be readily determined by in vitro binding or co-immunoprecipitation assays (see, eg, FIG. 3), where the targeting protein spectrin and / or (eg, ankyrin, protein 4.1, Addusin, see Table IB) Interaction with the adapter protein is evaluated. Such methods are well known in the art, as exemplified by Harlow et al., 1988.

For example, the interaction of Na, K-ATPase with 14-15 repeat units of spectrin is detected by co-precipitation (FIG. 3), and ankyrin binds to this region of spectrin ( Kennedy et al., 1991) and Na,
At a specific locus within K-ATPase (Devarajan et al., 1994), Na,
Via an ankyrin G119 adapter protein intermediate as detected by a direct binding assay showing that K-ATPase binds to ankyrin (Morrow et al., 1989).

Preparation of spectrin and associated cDNA constructs Using prokaryotic vector pGEX, recombinant peptides are prepared as fusion proteins with glutathione-S-transferase (Smith and Joh).
nson, 1998; Kenndy et al., 1991). As shown in FIG. 19, the spectrin loan used in these is βII spectrin (Hu et al., 1).
992; Chang et al., 1993; Chang and Forget, unpublished), βI spectrin (Winkelmann et al., 1988; Wink).
elmann et al., 1990b; Winradi et al., 1993), and β
Identical to the published sequence for III spectrin. Generally, the desired segment of the cDNA is excised by EcoRI and other appropriate restriction enzymes from pGEM7z (-), purified from an agarose gel by electrophoretic elution, and subcloned into an appropriate pGEX vector, usually pGEX-2T. Is done. In the example shown [FIG. 2 and (Kennedy et al., 1991; Kennedy et al., 1994; Lombardo et al., 1994b)], the polymerase chain reaction is used, typically in combination with Pfu polymerase and selected primers. Amplification of the region of the spectacle lawn allows for plasmid construction and, in some cases, linker-insertion mutagenesis (Sambrook et al., 198
9; Kennedy et al., 1991). In some cases, Taq
Using the polymerase, p of the TA Cloning Kit (Invitrogen)
It facilitated the cloning of the PCR product into the CRII vector. The validity of all PCR-generated constructs is confirmed by DNA sequencing. In general, it is useful, but not required, to create PCR primers 5 'to the BamHI site and 3' to the EcoRI site to facilitate directional subcloning into pGEX-2T. . Two to five additional bases were added to the end of each primer to enhance their sensitivity to restriction endonucleases. Amino acid boundaries for amplification of spectrin structural repeats are selected based on the fading of Drosophila α-spectrin (Winograd et al., 1991). Peptides representing the unique alternatively spliced COOH-terminal region of βI spectrin found in brain and muscle are prepared from plasmid pMSP6AC (Winkelmann, 1990) by digestion with XmaI. The resulting 767 bp fragment, containing the alternatively spliced unique C-terminus of spectrin βI ゲ ル II, is gel purified and subcloned into pGEX-2T at the SmaI site. Other typical details of primer selection and plasmid construction are summarized as in Kenedy et al., 1991; Lombardo et al., 1994b. Various βI spectrin peptides are expressed as described above from existing human βI spectrin constructs (W
Inkelmann et al., 1990a; Winkelmann et al., 1990.
b; Weed et al., 1996). Constructs for eukaryotic cell expression are typically
Prepared in pcDNA3-vector (Invitrogen) or other comparable vector incorporating a suitable eukaryotic promoter. Using the pcDNA3 vector, a stable MDCK cell line can be established by lipofection transfection (Weed et al., 1996) by selection with G418. Transient experiments can also be performed in this or some other system well suited for transient assays such as COS cells. In studies using VSV-G, newly confluent cells were infected with VSV-G for 60 minutes on a Transwell filter (100 ml, 108 titer virus Pimplikar et al., 19).
94). Infected cells are incubated for 3 hours and fixed prior to analysis.

Protein Expression and Purification The recombinant protein is expressed in E. coli strains HB101, DH5a or protease deficient strain CAG-456 and purified as described above (Smith and Joh).
nson, 1988; Kennedy et al., 1991). 1 if necessary
M NaBr, 10 mM Na ₂ HPO ₄ , 0.5 mM EGTA, 0.5 mM
In the presence of EDTA, 15 mM sodium pyrophosphate, 1 mM NaN ₃ , 0.5 mM DTT, pH 8.0, 100 of Sephacryl S-200 HR
Further purification by gel filtration on a cm column. 20m of purified protein
M Tris, 150mM NaCl, 1mM EDTA, 1mM NaN 3, 1m M DTT, 0.2mM PMSF, 25% glycerol, dialyzed into pH 7.5, and stored at -80 ° C.. Bovine brain spectrin is obtained by low ionic strength extraction of the decolonized brain membrane, followed by Sephacryl-S-500HR (Harris et al., 1985;
Bennett, 1986).

Antibodies and Immunodetection Procedure 8 aa A construct epitope tagged with a FLAG marker (IBI Scientific) was detected with Mab M5 (IBI Scientific); βI antibodies were Mab VIIIC7, Mab VD4, Mab IVC9, and Mab IVF8. (Harris et al., 1986); including Pab C19 (gift from V. Marchesi) for the 19COOH-terminal residue of βIｃｈ1 spectrin; and Pab MUSI (Malchiodi-Albedi et al., 1993) for region III of βIΣ2 spectrin. there were. P
ab 10D detected βII spectrin (Devarajan et al., 1996). Other antibodies are Mab β-COP, I.P. Melman; Pab E-Cadherin, Transduction labs; Mab α-Na, K-ATP
Mab β-Na, K-ATPase M.ase, UBI; Caplan;
Mab VSV-G, J.M. Rose; and Pab Centractin; H
olzbaur. Cells are grown on coverslips or culture dishes and PB
Washed three times with S, fixed in acetone for 15 minutes, blocked in goat serum for 30 minutes, and incubated with primary antibody in PBS + 2% BSA in 10% goat serum for 60 minutes at room temperature. All spectrin Mab were used at 1: 100;
1: 5000 E-cadherin; 1: 200 b-COP, 10D, C19. Detection used a secondary antibody conjugated to CY3 or CY2 (Vecto).
r).

The MDCK cells confluent with immunoprecipitation were washed with PBS, and then washed with an IP buffer (10 mM Tris-HCl, p
H7; 150 mM NaCl; 5 mM EDTA; 1 mM EGTA;
ml BSA; 0.5% deoxycholate; 1% NP-40; 0.5 mM Pefabloc; 1 mM PMSF; and 1 mM leupeptin) at 40C for 20 minutes by gentle rocking. Decant the lysate (2 ml),
Centrifuge at 10,000 xg for 1 minute and 200 ml of 50% protein ASepha.
6 with 25 ml of non-immune rabbit serum containing rose (Pharmacia).
Pre-cleared by incubating for 0 minutes. 200 remaining supernatant
ml of primary antibody at 4 ° for 4 hours, pelleted with Protein A Sepharose and analyzed.

Example 2: Na, K- as an essential membrane protein precipitated in SAATS
Identification of ATPase In wild-type MDCK cells, Golgi spectrin and Ank _G119 are localized in the Golgi, while α-Na, K-ATPase is predominantly distributed in the plasma membrane where it is the source of ankyrin. It is linked to the αIIβIII spectrum via the plasma membrane morphology (Morwo et al., 1989; Nelson and Ha).
mmerton, 1989) (FIG. 3).

To determine the subcellular distribution of Golgi spectrin, Ank _G119 and α-Na, K-ATPase, cells were grown on coverslips or culture dishes and PB
Washed three times with S, fixed in acetone for 15 minutes, blocked in goat serum for 30 minutes, and incubated with primary antibody in PBS + 2% BSA in 10% goat serum for 60 minutes at room temperature. Subcellular distribution of wt Golgi spectrin was determined by Mab
Monitored using VIIIC7. This is because of the shorter FLAG-tagged βI _N-5 construct (Harris et al., 1985; Devarajan et al., 1996; G
odi et al., 1997) but not with full-length Golgi spectrin. The distribution of βIΣ ^* spectrin in cells transfected with βI _{11 -C} was monitored by Mab VD4, which did not react with βI _{11 -C} . Antibody "Jasmin" (De
Ankara _G119 was monitored by Varajan et al., 1996), and α-Na,
K-ATPase is a Mab from Upstate Biotechnology
Monitored by Detection was by indirect immunofluorescence.

Electron microscopy was performed by fixing the cell monolayer in situ for 1 hour (Karnovsky's fixative 18). 0.1M fixed monolayer
Na-cacodylate, washed with pH 7.4, 1% O in 0.1 Ms-collidine
and post-fixed in sO _4. After dehydration in graded ethanol and washing with propylene oxide, samples were washed with Epox-812 (Ernest Fullman,
Inc. N. Y. ). Ultrathin sections stained with aqueous solutions of uranyl acetate and lead citrate were observed on a Zeiss EM-910 at 80 kV.

In FIG. 3A, the results of the indirect-immunofluorescent labeling are presented. _Note the dispersion of endogenous Golgi spectrin, Ank _G119 and α-Na, K-ATPase by βI _N-5 (but not βI _11-C ). All shortened spectrin constructs containing region I and MAD1 exhibit similar effects, while constructs lacking these regions of βI spectrin show βIΣ ^* spectrin, _AnkG119 or Na, K- It did not significantly alter the natural distribution of ATPase. Bar = 10μ

[0059] Endogenous Golgi spectrin-ankyrin and Na, K-ATPase
The distribution of the membrane assembly of was disrupted by the βI spectrin construct containing the region I / MAD1 Golgi targeting signal (FIG. 3A). In the presence of such peptides, endogenous Golgi βIΣ ^* spectrin and Ank _G119 were distributed in coarse punctate sol patches (FIGS. 3A and 4A). More dramatic is the effect of these peptides on the distribution of Na, K-ATPase, which becomes widely distributed throughout the cytoplasm in a pattern similar to the endoplasmic reticulum, with very few proteins identifiable at the cell surface ( (FIGS. 3A, 4A). Compared to control cells, βIN _-5 expressing cells are swollen (mean diameter 29 ± 6.7 m vs. 17 ± 3.7 m) and probably have insufficient plasma membrane Na, K-ATPase (or other membranes). Grow slowly due to protein (see below). This effect on the Na, K-ATPase distribution is due to the region I / M
Constructs lacking AD1 (eg, βI _11-C , FIG. 3A) are not evident and contain full-length βIΣ1 spectrin (containing ankyrin-Na, K-ATPase binding domain but lacking a PtdInsP ₂ -dependent Golgi bond) It is not clear even in cells expressing.

Co-immunoprecipitation experiments are performed to determine the average value of the interaction between Na, K-ATPase and spectrin. Briefly, confluent MDCK cells were washed with PBS and washed with IP buffer (10 mM Tris-HCl, pH 7; 150 mM NaCl; 5
1 mM EGTA; 2 mg / ml BSA; 0.5% deoxycholate; 1% NP-40; 0.5 mM pefablock; 1 mM PMSF; and 1 mM leupeptin) by gentle rocking to 4 ° C. To dissolve for 20 minutes. The lysate (2 ml) was decanted, centrifuged at 10,000 xg for 1 minute, and 200 ml of 50% protein ASepharose (Pharmaci).
Precleared by incubating with 25 ml of non-immune rabbit serum containing a) for 60 minutes. The remaining supernatant was treated with 200 ml of primary antibody at 4 ° for 4 hours, pelleted with protein ASepharose, and SDS-P
After AGE, they were analyzed by Western blot and visualized by ECL. Co-immunoprecipitation experiments show that the interaction of Na, K-ATPase with the 14-15 repeat units of spectrin occurs via an ankyrin G119 adapter protein intermediate (see Figure 3b). In wt cells, Betaaishiguma ^* spectrin, An k _G119, St. Rakuchin, and Na, K-ATPase is coprecipitated from detergent lysates by Mab VIIIC7 (lane IP). Soluble αIIβII spectrin (detected here by immunoblotting for αII spectrin), a form predominantly found in the plasma membrane, is also present in the detergent lysate (
Lane lys), not part of the precipitable Golgi spectrin complex. Control experiments with pre-immune or irrelevant antibodies did not precipitate any of these components. Similarly, immunoprecipitation of βIN _-5 transfected cells with Mab VIIIC7 also showed co-precipitation of _AnkG119 and Na, K-ATPase with βIΣ ^* spectrin, which was induced by the βIN _-5 construct. Despite the dispersal of these elements, they show that they remain associated in the detergent lysate.

As illustrated by FIG. 3 b, immunoprecipitation with Mab VIIIC7 shows that α-Na, K-ATPase fractions are present in a complex with βIΣ ^* spectrin, Ank _G119 and _centlactin (FIG. 3 b). Centracin (AR
P-1) is an actin-related protein that is part of the dynactin complex.
This pool of α-Na, K-ATPase associated with βIΣ ^* spectrin, Ank _G119 and _{centracin appears} to be distinct from the pool of plasma membrane α-Na, K-ATPase. This is because not all spectrin is present in these complexes (FIG. 3B) and probably Na not yet assembled at the plasma membrane.
, K-ATPase.

[0062] Na, K-ATPase is blocked by the ER against medial-Golgi movement by βIN _-5 spectrin.

Usually, after synthesis and core glycosylation at the ER, β-Na, K-ATPase undergoes further glycosylation at the medial-Golgi, followed by α, β-Na, K-AT
Vector-incorporated as detergent homodimers into detergent-insoluble αIIβII spectrin and ankyrin cortical skeletal lattices (Nelson and Veshnoc
k, 1986; Morrow et al., 1989; Nelson and Ham.
merton, 1989; Mays et al., 1995). The distribution of Na, K-ATPase in βIN _-5 expressing cells suggests that Na, K-ATPase escape from the ER may be blocked (FIG. 4A).

Western blotting confirmed that the spectrin βIN _-5 polypeptide blocked the Golgi-mediated glycosylation of β-Na, K-ATPase (FIG. 4b).
. Control MDCK cells (wt) or cells transfected with the βIN _-5 construct (βIN _-5 ) were analyzed by Western blot using a β-Na, K-ATPase specific antibody (Mab B1-13). . The apparent MW of various products is shown. 50 kDa (gly representing mature glycosylated β-Na, K-ATPase
Note the broad band above) and the strong band at 44 kDa in βIN _-5 cells (core) representing the ER-dependent core β-Na, K-ATPase glycosylation product. Thus, βIN _-5 spectrin peptide inhibits the mature glycosylation of β-Na, K-ATPase, a process characteristic of medial-Golgi processing, but interferes with the formation of core glycosylation products that occur in the ER. Seems not. In this experiment, as judged by immunofluorescence, approximately half of the beta I _N-5 cells express beta I _{N -5} construct. It appears to account for most of the mature glycosylation products observed in the βIN _-5 cell line. Band in wild type cells (*) = 106 kD
a, β-Na, K-ATPas, as its appearance is variable and is usually observed in MDCK cells after reduced SDS-PAGE (Morrow et al., 1989).
e appears to represent a cross-linking adduct with α-Na, K-ATPase or other proteins.

Although these experiments do not require a quantitative measure of the extent of transport inhibition, the results of some other measurements indicate that β-Na, K-A TPase by βIN _-5 peptide It shows that the efficiency of transport inhibition is quite high. This is because detectable levels of processed β-Na, K-ATPase are inversely correlated with the fraction of cells in those cultures that actually express the recombinant peptide. Also blocked in these cells was the translocation of α-Na, K-ATPase to detergent insolubility (FIG. 4C), which was associated with a stable plasma membrane associated spectrin lattice in which Na, K-ATPase was stable. Indicates not to join. Α- after SDS-PAGE
A Western blot for Na, K-ATPase is shown. Fx1 (soluble fraction) is material solubilized by 0.5% Triton X-100 in 100 mM NaCl; Fx2 (cytoskeletal fraction) is 0.5% Triton X-10.
0 and 250 mM NH ₄ SO ₄ (Devarajan et al., 1994). The presence of a βI _11-5 polypeptide containing this competent ankyrin-binding domain but lacking the region I / MAD1 sequence (comparison, FIG. 2C
), Only slightly impacted the assembly of Na, K-ATPase into the detergent-insoluble skeleton. Taken together, these data indicate that a truncated βI spectrin peptide containing the Golgi targeting signal but lacking downstream sequences including the ankyrin / Na, K-ATPase binding domain has both Na and K-ATPase subunits. E
Indicates that transport from R to the medial-Golgi is blocked.

The interaction of SAATS with Na, K-ATPase occurs via an adapter protein. As described above, co-precipitation of wild-type cells with antibodies to βI spectrin
Precipitate a complex containing spectrin, _AnkG119 , _centlactin and Na, K-ATPase (FIG. 3). Independent experiments using an in vitro assay show that α-Na, K-ATPase binds directly to ankyrin (Morro
W et al., 1989; Devarajan et al., 1994), Na, K-ATP.
It shows that a small 25 residue region of ase, codons 142-166, accounts for most of this binding activity (for the minimal ankyrin binding domain, this region is MA
B) (FIG. 8) (Zhang et al., 1997a). Other experiments have established that ankyrin binds to the fifteenth repeat unit of spectrin (Kennedy et al., 1991).

The requirement that ankyrin binds to SAATS in Na, K-ATPase transport is therefore: i) if the ankyrin binding domain is otherwise Na, K-ATPase
beta it _N-5 is returned to beta I _N-5 peptide to block the SAATS mediated transport of se, ₁₅ spectrin construct in cells responsible for (FIG. 2), Na to the plasma membrane, K-AT
By demonstrating that the ankyrin-binding domain of spectrin is required for transport, as evidenced by the rescue of Pase delivery (FIG. 6); and Na, K
-Demonstration by showing that removal of the specific ankyrin binding sequence from ATPase renders the protein unable to transport from the ER (Figure 9). Further evidence of the importance of this binding to the adapter protein is that (in this case) its transport by expression (or microinjection) of a specific inhibitory peptide that blocks the interaction of α-Na, K-ATPase with ankyrin. It can also be obtained by selective inhibition (FIGS. 10, 18).

[0068] In FIG. 6, in beta I _N-5 MDCK cells as described above, or transfected with beta I _N-5,15. This latter construct incorporates the ankyrin-binding domain of spectrin into the βIN _-5 peptide. In this case it is measured by its surface display and secondarily by a decrease in cell size (resulting from cell swelling due to lack of plasma membrane Na, K-ATPase in βIN _-5 transfected cells). Note the complete recovery of Na, K-ATPase transport.

In FIG. 9, a FLAG epitope tag was added at its NH2 terminus to wild-type Na, K-ATPase, or a similarly FLAG-tagged mutant Na, K- deleted with codons 142-166 deleted. By ATPase, MDC
K cells were transiently transfected. These residues correspond to the minimal ankyrin binding domain (MAB) identified in FIG. In both transfections, cells were co-transfected with wt β-Na, K-ATPase to ensure that sufficient β-Na, K-ATPase matched α-Na, K-ATPase. (Top) FLAG-tagged wt-Na, K-ATPase is normally delivered to the plasma membrane. (Bottom) FLAG-tagged Na, K-ATP lacking MAB
ase is not assembled at the plasma membrane and is eventually targeted for degradation in the lysosome. In these cells, this is the only protein whose transport is apparently disrupted.

Example 3 Identification of VSV-G as an Essential Membrane Protein That Participates in SAATS
Coat (for review, see Skekman and Orci, 1996)
VSV-G transport to the plasma membrane, which is exclusively packed by, was shown to be blocked by the βIN _-5 spectrin construct (FIGS. 5E, F). V observed
The pattern of SV-G staining suggests a block prior to the middle compartment. Wild type (B
, D, F, H) and βIN _-5 transfected cells (A, C, E, G). Localization of VSV-G protein (E, F) was measured after transient infection.
E-Cadherin (C, D) was prepared from Ma from Transduction labs.
b, the extent to which the precursor peptide was degraded from 135 kDa ( ^* ) to 120 kDa (EC) (inset, Western blot) (trans-Golgi / S
hole and Nelson, 1991). There was no significant difference in the degree of processing of E-cadherin in the βIN _-5 line vs. wt cells or its level of assembly at the plasma membrane. Electron microscope stained by the distribution of β-COP (A, B) and uranyl acetate and lead stained despite disruption of Na, K-ATPase and VSV-G transport (and wt Golgi spectrin skeleton, FIG. 3) The Golgi looks fairly intact, as measured by the presence of a normally visible proximal nuclear Golgi structure in the observations (G, H) (arrows). Bar = (A-
10μ in F) and 0.5μ in (G, H). Original magnification (G, H
) = 63,000 ×.

Unlike α-Na, K-ATPase, VSV-, as indicated by the inability of the specky ankyrin-binding domain (repeat 15) to be rescued by reversion to the inhibitory βIN _-5 construct, is shown. The G protein is transported by SAATS without using ankyrin as its adapter protein (FIG. 7).
MDCK cells infected with vesicular stomatitis virus were used to monitor VSV-G protein transport. The newly confluent cells are infected with a Transwell filter (100 ml, 108 titer virus Pompirokar et al., 1994) for 60 minutes. Infected cells are incubated for 3 hours and fixed prior to analysis. After 60 minutes blocking with goat antiserum, 60 minutes at room temperature,
Mab VSV-G (J. Ross) in 2% BSA in PBS + 10% goat serum
e) was used as the primary antibody. Similar to that of Na, K-ATPase (in wt cells and as shown in FIG. 5), the transport of this protein
Usually to the basolateral membrane. This transport is based on Na, K-ATPase
As shown, it is blocked by SAATS inhibition with the βIN _-5 construct (comparison, FIG. 6). However, unlike Na, K-ATPase, VSV-G transport is not restored by including the ankyrin binding domain of spectrin (repeats 14-15) in the construct. In this way, it is possible to selectively block VSV-G transport without affecting Na, K-ATPase transport.

Example 4: Determination that E-cadherin sequestration is not blocked by βIN _-5 spectrin E-cadherin, another type I basolateral targeting membrane protein, was expressed by SAA
It was assayed for its interaction with TS. Wild type (FIG. 5D) and βIN _-5 transfected cells (FIG. 5C) were assayed for E-cadherin distribution. Three
After blocking with goat antiserum for 0 minutes, PBS + 10% for 60 minutes at room temperature
Transduction lab as primary antibody in 2% BSA in goat serum
E-cadherin (C, D) was monitored with the Mab from S.
*) Degree of proteolysis of precursor peptide from 120 kDa (EC) (inset, western blot) (process occurring in trans-Golgi, Shore
And Nelson, 1991). There was no significant difference in the degree of processing of E-cadherin and the degree of assembly at the plasma membrane in βIN _-5 vs. WT cells. Na, K-ATPase and VS
Despite disruption of VG transport (and wt Golgi spectrin skeleton, FIG. 3), by distribution of β-COP (A, B), and by uranyl acetate and lead staining molecular microscopy (G, H) ( The Golgi appears largely intact, as measured by the presence of a normally appearing proximate Golgi structure in arrow). Bar = (A-
F): 10μ, (G, H): 0.5μ. Original magnification (G, H) = 63,0
00x was well expressed at the plasma membrane (FIGS. 5C, D).

[0073] Western blot analysis of the processing of E-cadherin, including protease cleavage in the trans-Golgi compartment, where the precursor protein is reduced from 135 to 120 kDa (Shore and Nelson, 1991) showed that the uncleaved E-
No increase in cadherin pool could be detected (FIG. 5D, inset). Thus, unlike Na, K-ATPase and VSV-G, the transport of E-cadherin is not blocked in the ER to Golgi transition, and its attachment to SAATS occurs within the βIN _-5 peptide, Or indicate that it could be assembled by another route otherwise.

Example 5 α5 as an Essential Membrane Protein That Participates in SAATS in Endothelial Cells
Identification of αV, β3 and βI integrins A rapid method for assessing the involvement of various essential membrane proteins in SAATS delivery is their use in cells transfected with the various constructs shown in FIG. 2 or related constructs. The use of flow cytometry to monitor surface displays. Examples of this approach are shown in FIGS. FIG.
In 4, another somewhat worse blocker of SAATS function (βIN _-2 peptide) was used with comparable results.

In FIG. 13, SV40 transformed murine endothelial cells were transfected with βIN _-5 or βIN _-5,15 and surface display of three different integrins was measured by flow cytometry. Note the blockade of alphaV, beta3 and beta1 integrins by the βIN _-5 peptide. Conversely, the βIN _-5,15 peptide significantly enhances the surface display of alpha V and beta 1 integrins (but not beta 3 integrins). This enhanced display (as opposed to blocking) may increase the efficiency of SAATS-directed loading of vesicle transport, possibly by selecting peptides or agents that enhance the binding of a given membrane protein to SAATS. Achieved by augmentation.

In FIG. 14, the experiment was performed as shown in FIG. SV40-transformed murine endothelial cells were transfected with βIN _-5 , βIN _-5,15 or βIN _-2,15 , and the display of three different integrins was measured by flow cytometry.
Note that alpha-V integrins are also modulated by SAATS. Also note that in this example, βIN _-2,15 was used to rescue beta-I expression. Although βI _N-5 is a more broadly inhibitor of SAATS transport (see FIG. 1), in this case, the supernormal levels of beta-I include the inclusion of repeats 14,15 in the βI _N-5 peptide. Can be achieved by:

Example 6: Identification of Other Proteins That Participate in SAATS Transport in Lymphocytes Using flow cytometry in combination with the methods outlined here, it is possible to identify other proteins as modulated by SAATS. Yes, and those modulated by the adapter protein ankyrin are also evident. Only those that were modulated by ankyrin were repeated 14,1 in the inhibitory spectrin construct.
5 would be saved. In FIG. 15, PE
CAM (CD31) is shown to be ankyrin / SAATS transported in lymphocytes. Cells are transfected as described above, PECAM, IgG
Superfamily intercellular adhesion molecules were monitored by flow cytometry. For both constructs, the inclusion of the βIN _-5 sequence resulted in a strong blockade of transport, indicating their involvement with SAATS. In both cases, the surface display was rescued or complemented by the βIN _-5,15 peptide. This effect is likely due to the interaction of GFP with the COOH-terminal 15th repeat until the SAATS binding activity to PECAM needs to be restored, resulting in an N-terminal FL.
AG-tagged versus COOH-terminal GFP-tagged constructs were somewhat prominent.

In FIG. 16, CD45 is shown to be a SAATS regulated by its putative adapter, ankyrin, while TNFR-1 is modulated by SAATS but does not include ankyrin as its adapter molecule. In this experiment, Jurkat T-lymphocytes were transfected with the indicated constructs, including βI _14-15 alone. Note the marked down-regulation of CD45 (ankyrin binding protein described) by the construct lacking repeat 15 and by the βI _14-15 peptide (lacking the constitutive Golgi targeting signal (see FIG. 2)). I want to. Also,
These experiments show that TNFR-1 display is not affected by βIN _-2 or βIN _-5 (
(Rather than blocked), indicating that, like E-cadherin, attachment to SAATS is mediated directly or indirectly by sequences contained within βIN _-2 . Suggest that

In FIG. 17, Fas and Fas-L are shown to be SAATS-dependent, but only Fas appears to utilize ankyrin. Jurkat lymphocytes were transfected with the indicated constructs. The experiment was performed as described above. Note the changes in both Fas and Fas-L by βIN _-5 and βIN _-5,15 peptides.

Example 7: Selective Inhibition of Single Protein Transport By utilizing a specific inhibitor of docking of a single essential membrane protein to SAATS, a more effective blocker of SAATS function than Rather, great specificity has been achieved. For example, the ankyrin-binding droin sequence of α-Na, K-ATPase is highly conserved across all species of Na, K-ATPase, but is not found in any other protein (Zhang et al., 199).
7b). Expression of this peptide sequence alone in cells (FIG. 10) selectively blocks the transport of Na, K-ATPase without blocking the transport of any other known protein. Thus, great specificity can be achieved by the identification of selective inhibitors of proteins that dock to SAATS.

In FIG. 10, wild-type MDCK cells were transiently transfected with a green fluorescent protein linked to a 25 residue MAB peptide. This means that the deletion is N
a, K-ATPase is the same sequence that allows the ER and eventually the lysosome to be retained in FIG. After culturing for approximately 1-2 days, cells were fixed and (left panel using anti-GFP antibody) per GFP or Na, K-ATPas.
e (right panel, red). GEP in MDCK cells (wt)
Expression alone is based on Na, K-ATPase distribution or cell size (this is
-Dependent on the ATPase function). However, GEP is 2
When a 5-residue MAB peptide is carried, endogenous Na, K-
ATPase binding was inhibited, resulting in selective accumulation in the ER and reduced levels on the plasma membrane (two examples are shown, labeled "MAB"). Also, GF
Cells containing P-MAB swell significantly, as expected. Other protein distributions were not affected in these cells.

In FIG. 18, clusters of wild-type MDCK cells were microinjected with (A) GST alone or (B) βIN _-4 peptide generated as a recombinant fusion peptide with GST, followed by indirect Stained for Na, K-ATPase by immunofluorescence. Microinjected GST had no effect, but (B) NST in the injected cells on the left
a, Note the loss of K-ATPase staining intensity. Impaired Na to the membrane,
The results of K-ATPase delivery are evident in the larger size of cells with insufficient Na, K-ATPase, as well as the changes observed when transfecting βIN _-5 . These results indicate that selective blockade of Na, K-ATPase can be achieved by exogenous small peptides. Similar experiments were performed using various monoclonal antibodies. In these experiments, cells were in confluence and all cells in the isolated cluster were injected. (C) The buffer alone control has no effect on the distribution of Na, K-ATPase. (D) (not considered to be a component of SAATS) Microinjection of Mab IID2, which reacts exclusively with aI spectrin, also has no effect on Na, K-ATPase.
(E) Reacts with βI spectrin (Harris et al., 1986) Mab V
Microinjection of IIIC7 leads to severe disruption of Na, K-ATPase delivery, accumulating considerably within cells and swelling cells. In this micrograph, the loss of the membrane Na, K-ATPase is quite severe and the membrane boundaries cannot be recognized.
This experiment demonstrates the effect of SAATS-specific monoclonal antibodies in blocking SAATS activity.

Example 8 Cystic Fibrosis Transmembrane Conduction Regulators Participate in SAATS Transport FIG. 12 shows another essential membrane protein of considerable medical interest linked to SAATS by different ankyrin interactions Is shown. (A) CFTR (cystic fibrosis transmembrane conductivity regulator) and its closely homologous TNR (transmembrane-
Nucleotide binding-regulatory domain (Transmembrane-Nuclee)
The oide binding-Regulatory domain protein is present in the soluble (Fx1) and cytoskeletal pool (Fx2) of confluent MDCK cells. These cells have a tip surface (lane A) or basolateral surface (lane B)
From either, when surface labeled with biotin, both CFTR and TNR are highly polarized and, unlike Na, K-ATPase, are predominantly expressed in the apical domain of these polarized epithelial cells. (B) Despite the fact that CFTR and TNR are tip proteins, they nevertheless bind to components of the SAATS system that are responsible for their transport. Either RIPA buffer or whole cell lysates of cells extracted with Fx1 and Fx2 (L) were used as irrelevant antibody controls (C)
Alternatively, immunoprecipitation was performed with an anti-CFTR antibody (IP). The washed immunoprecipitates were then analyzed by SDS-PAGE and examined by Western blotting for either Ank _G119 or ANK _R. Na, K-ATPase is SAAT
Just as it binds to S, both ankyrins co-precipitate with CFTR. However, CFTR and Na, K-ATPase bind at different sites on SAATS, allowing their selective modulation.

FIG. 22 shows cystic fibrosis transmembrane conductivity regulator (CF) by SAATS.
13 shows the modulation of the surface display of TR). MDCK cells were transfected with beta I _N-5 or beta I _{N-5, 15} construct, then surface labeled with biotin. After labeling, cells were lysed and all CFTR in each culture was immunoprecipitated with anti-CFTR antibody. The precipitate was then analyzed by SDS-PAGE, transferred to nitrocellulose, and surface CFTR was detected by avidin labeling. Also, total CFTR was measured in those cell extracts by Western blotting (data not shown). In all cell lines, there was minimal change in total CFTR in the cells. However, there was a marked decrease in the surface display of CFTR induced by the SAATS blocker βIN _-5 ; whereas, the βIN _-5,15 construct significantly enhanced the surface display of CFTR. Taken together, these results describe the involvement of SAATS in CFTR delivery and describe a possible therapeutic approach to the control of disorders such as cystic fibrosis, or related proteins such as the multidrug resistance gene product (MDR). Suggest.

Example 9 Selective Enhancement of Essential Membrane Protein Display by SAATS The rate of exit of essential membrane and secretory proteins from the ER and Golgi is at least partially due to their rate and efficiency of docking to SAATS. Seems to be dynamically adjusted. Thus, the up-regulation of surface display or secretion of a given protein, as well as its blocking,
This can be achieved by modulation of the ATS. This is shown in FIGS. 13-16, where for molecules utilizing ankyrin as an adapter complex, those with βIN _-5,15 peptides that increase ankyrin-dependent transport at the expense of ankyrin-independent transport. Ultranormal levels are achieved by increasing the transport of
For example, TNFR-1 depression in βIN _-5,15 expression, FIG. 16).

Example 10: Determining which vesicle backbone proteins and associated adapter proteins are specifically involved in mediating sequestration of membranes or secreted proteins of a given cell into transport vesicles The general method described here is that the delivery of a given membrane protein or a class of membrane proteins to the plasma membrane of a eukaryotic cell can be accomplished by using such a protein in a spectrin / ankyrin / adaptor protein delivery system ( (SAATS) can be inhibited or increased by altering binding. The secretion of proteins that bind to so-called luggage receptors in the endoplasmic reticulum can also be controlled by controlling the binding of such luggage receptor proteins to SAATS.

As disclosed herein, SAATS act to concentrate proteins specific to the endoplasmic reticulum (ER) and facilitate their inclusion in vesicles that undergo transport or diffusion between the ER and the Golgi compartment. Latent protein sorting device. Although a similar pathway is active between the Golgi and the plasma membrane of many cells, the most common use of this method of control is achieved by blocking SAATS in ER or cis Golgi relocation. The method comprises: 1) identifying which component of the SAATS system binds a given essential membrane protein, as described below, and then 2) disrupting the binding interaction between the target protein and SAATS in vivo. This is done by identifying peptides, small molecule analogs, antibodies, or antisense oligonucleotides that will do so. This latter aspect of the invention is discussed in the next section of this specification.

Previous efforts have only identified binding sites for a small number of those we know to be SAATS ligands today (see Table 1B). We believe this is due to the fact that many interactions will occur in solutions with only low affinity, multiple affinities that will be significant in the restricted environment of SAATS-membrane interactions. Specifically, once the entropic contribution to binding energy is accommodated by high affinity SAATS-membrane association (as through MAD1 or MAD2) (Lombardo et al., 1994), other interactions between membrane proteins and SAATS Will be promoted. Assays for detecting such interactions must therefore be sensitive to detecting low affinity but specific interactions.

The standard techniques for measuring protein-protein interactions at equilibrium are thus typically weak interactions that can characterize binary interactions between membrane proteins and many ligand sites in SAATS (Ka = 10 ^-4 to 10 ^-5 ). Typical of such assays are equilibrium dialysis; fluorescence-based assays that monitor the change in the intrinsic fluorescence of the protein as a function of binding; assays based on osmotic pressure or light scatter; commonly known to those skilled in the art. Would have been. In addition, Pharmacia Bio
Surface plasmon resonance, such as that performed on the Core instrument, is also suitable for detecting specific macromolecular interactions within SAATS, often even in crude solutions prepared from cell or bacterial lysates. Other methods of enhancing detection sensitivity are by using radioactively labeled compounds or other sensitive detection techniques such as enhanced chemiluminescence in combination with standard microtiter or blot overlay techniques. Two-hybrid assays in ye
Gene selection assays such as ast) or epitope display library screening can also be used.

Example 11: Identification of peptides and small molecules that modulate sequestration and / or vesicle trafficking of essential membrane and secretory proteins In general, a number of approaches have been used between the target protein and the SAATS. Can be identified that can block (or increase) the interaction of. In general,
This method comprises the steps of: A) disrupting a class or multiple classes of membrane or secreted proteins, such as a peptide drug or an expressed recombinant gene encoding a particular functional domain in SAATS, By identifying inhibitors with broad SAATS function that inhibit the docking of multiple classes of membrane proteins by competitive inhibition with, or B) based on detailed knowledge of the binding sites that mediate the attachment of target proteins to SAATS Approaches can be made through drug design strategies that can be either rational with inhibitory agents determined or random based on in vitro assay surrogate assays of SAATS function. The latter approach is based on identifying compounds that block interaction with SAATS as measured by an in vitro binding assay.

Examples of approach A) have been presented in section A of the detailed description above. Examples utilizing approach B are outlined below, along with a general protocol for identifying such agents.

B) Strategies based on rational drug design. Blocking or modulating the display of membrane protein X using a rational drug design approach would require detailed knowledge of its SAATS binding site, up to and including its three-dimensional structure. Completion of steps A4 and A5 provides a starting point for a systematic approach.

Step B1: When mature, determine whether protein X is a monomer or a homopolymer, or whether it is present in its mature state as a heterocomplex with another membrane protein. If protein X is shown to bind directly to SAATS, and many membrane proteins and all secreted proteins only attach to SAATS through their interaction with another membrane protein, This step would not be necessary. If this were the case, drugs that would prevent heterocomplex formation would have to be identified,
Alternatively, the complex will be controlled because the units (ie, the display of all components of the functional heterocomplex) will be controlled by controlling their SAATS assembly. These determinations can be made using standard protein biochemical and biophysical techniques, including co-immunoprecipitation from non-ionic detergent extracts of cultured cells expressing protein X.

For example, α-Na, K-ATPase (as opposed to β-Na, K-ATPase)
Demonstration of direct binding of se to ankyrin (Morrow et al., 1989, De.
varanjan et al., 1994) can be made by a variety of in vitro and in vivo techniques, including coprecipitation, gel overlay binding assays, and surface plasmon resonance. In these assays (eg, Morro
et al., 1989), α-Na, K instead of β-Na, K-ATPase.
-Can demonstrate direct binding of ankyrin to ATPase. However, while β-Na, K-ATPase binds stoichiometrically to the alpha subunit, both are well transported by SAATS, but this transport is due to α-Na, K
-Depends on the binding of ATPase to SAATS (eg, β-Na, K-AT
(See FIG. 4 showing that Pase transport is not possible.)

Step B2: Determination of a specific binding site within Protein X that links it to SAATS. This can be accomplished using a number of conventional methods. The usual ones are
The use of deletion mutagenesis with a series of recombinant peptides, often produced as fusion peptides linked to glutathione-S-transferase (GST). For example, deletion analysis of a GST-fusion peptide representing the cytoplasmic domain of Na, K-ATPase (Devaranjan et al., 1994a; Devaran
jan et al., 1994b).

(Proceed to step B3 or an alternative step to B3) Step B3: Use of a conventional structure determination method to analyze the 3-D structure of the binding domain. If complete structure determination of protein X is not possible, a useful method for determining the relevant structure of the binding site is carrier-mediated crystallization, where the binding domain of interest crystallizes as a fusion protein with GST. (For example, Lim
Et al., 1994). Subsequent determination of the structure of this complex gives the structure of the key binding site. Other methods for determining the structure, such as multidimensional NMR, can of course apply as well.

For example, α-Na, K-ATPas by crystallization of active GST-fusion peptide
Determination of the minimal ankyrin binding domain in e will be useful as we have recently shown (Zhang et al., 1997b). See FIGS. 4b, 4C and 11. To demonstrate the efficacy of drugs based on structures such as the binding domain (FIG. 10)
SAAT of the native protein using the binding domain alone)
It is also useful to demonstrate the specific inhibition of S transport and the ability of the binding domain to bind in vitro to its ligand receptor site with affinity and stoichiometry comparable to that of the native protein.

Step B4: Design assays for candidate small molecules that will inhibit binding interactions and those compounds with their ability to block the in vitro interaction between Protein X and its SAATS binding site Use of the decision structure for: Alternatively, if the target identifies a small molecule that increases a given SAATS interaction, it may be desirable to treat a child with cystic fibrosis (FIG. 12B) and display a given protein. Or to identify therapeutic agents that enhance transport or secretion, molecules that increase the in vitro interaction between Protein X and SAATS would be needed.

Step B3 Alternative: Alternatively, an empirical approach to identifying lead compounds based on alternative measurements of SAATS function is possible. Since all or most proteins exit the ER compartment via direct or indirect binding to SAATS, in vitro assays based on specific binding sites for each membrane or secreted protein of interest will require SAATS function. Provides an ideal basis for high-throughput in vitro screening. For example, high-throughput screening in microtiter plates, seeking to identify all agents that block or reduce the binding of spectrin to Ank _G119 , or Ank _G119 to protein X, is one such suitable Would be an assay. Alternatively, protein X or ankyrin or other SAATS components are immobilized on an inert substrate (eg, nitrocellulose, immobilon, 96-well titer plate, microchip, etc.) and various compounds are immobilized on the immobilized protein. A similar assay could be prepared that compares for their ability to block or enhance the binding of soluble proteins to E. coli. The details of performing such assays are well known in the art. A key feature of these assays applied to SAATS is that direct binary interactions between essential membrane proteins, or partially purified oligomeric complexes of such proteins, are required for drug discovery. Can be used as a target. I) such proteins can be immobilized even in impure form; ii) the purified and well-characterized components of SAATS are capable of binding to such immobilized or impure proteins. Can be monitored; III) The presence of a binary interaction is itself a well-proven good alternative for in vivo SAATS activity.

Example 12: Strategy based on the regulation of spectrin binding to the Golgi complex We have determined that the rate of specific secretion and transport of essential membrane proteins from the ER to the plasma membrane is , Would be prepared by controlling binding to SAATS. Several levels of post-translational regulation of the spectrin cortical cytoskeleton have been identified, and we predict that some of these are active in regulating SAATS (Morrow et al., 1997). . In addition, the regulation of SAATS regulates the assembly of other coretamer components in the secretory pathway and is a modulation of SAATS affinity for the Golgi by ADP-ribosylation factor (ARF) (Go
di et al., 1997) appear to possess at least one regulatory pathway.

In one embodiment of the invention, to assess the activity of a candidate compound to modulate the interaction of SAATS with a selected essential membrane or secreted protein,
Such a compound is mixed with the selected protein, and any vesicle scaffold protein or adapter protein is involved. After mixing under conditions that allow the association of these components, the mixture is analyzed and the drug is tested for SAAT of the selected protein.
Determine whether binding to the S binding partner was inhibited or enhanced.

Thus, inhibitory and potentiating agents are identified as being able to modulate this interaction. One of skill in the art would readily employ a number of techniques known in the art to determine whether a particular agent modulates the binding of such a selected protein to a SAATS binding partner. can do. The agent can be further tested for its ability to modulate binding using a cell-free or cellular assay system. Example 14 provides one such method that can be used to assay for related activity.

As used herein, an agent is an agent that reduces binding of a selected essential membrane or secreted protein to its SAATS binding partner,
It is said to inhibit AATS binding activity. Preferred inhibitory agents specifically and selectively inhibit such binding and do not affect the interaction of any other protein with SAATS. Further, preferred inhibitory agents are greater than 50%, more preferably about 9%.
Such binding is reduced by more than 0%, and most preferably substantially eliminates all binding of the selected protein to SAATS.

As used herein, an agent is an SAAT of an essential membrane protein from which the agent is selected.
When increasing the binding to S, it is said that SAATS binding activity is enhanced. Preferred binding enhancing agents are those that have greater than about 50%, more preferably greater than about 90%, SAA
It increases TS binding activity and most preferably doubles the level of binding or transport of such proteins to SAATS.

[0105] Preferred inhibitory and potentiating agents will be selective for a particular species of essential membrane or secreted protein. The agents of the present invention can be, by way of example, peptides, small molecules, and vitamin derivatives, and carbohydrates. One skilled in the art will recognize that there are no limitations as to the structural properties of the agents of the present invention. One class of agents of the invention is selected for its amino acid sequence based on the amino acid sequences of various spectrin analogs. Small peptide drugs can serve as competitive inhibitors of SAATS binding, sequestration and transport.

The peptide agents of the present invention can be prepared using standard solid phase (or liquid phase) peptide synthesis methods, as known in the art. In addition, DNAs encoding these peptides can be synthesized using commercially available oligonucleotide synthesizers and can be produced recombinantly using standard production systems. Production using solid phase peptide synthesis is required when non-gene encoded amino acids are to be included.

Another class of agents of the invention are antibodies that are immunoreactive with key epitopes of vesicle skeletal proteins such as spectrin and associated adapters such as ankyrin. Antibodies are obtained by immunizing a suitable mammalian subject with a peptide that binds to an antigenic region, selected membranes or secretes, eg, contains spectrin. Key regions for the transport of Na, K-ATPase include the domain identified in FIG. 3 or the cytoplasmic 29 residues of VSV-G.
Such agents can be used in competitive binding experiments to identify second generation inhibitors.

Example 13 Use of Agents that Modulate Sequestration and / or Vesicle Transport of Essential Membrane and Secretory Proteins for Therapeutic Purposes We have found that the method is useful in a wide variety of therapeutic senses. I believe that. For example, Table 2 below shows some possibilities.

Table 2 Possible therapeutics for the following diseases

[0110]

The administration of an agent as identified by the above-described techniques may be intended for its intended purpose, ie, to enhance or inhibit the transport of selected essential membrane or secreted proteins, and its chemistry, ie, peptide or Will depend on the small molecule. For example, to treat cystic fibrosis, as described above,
Identifying the AATS-binding domain is a function of the mutant (but functional) CF
It will allow the identification of agents that selectively increase the delivery of TR to the plasma membrane. Suitable agents will optimally be delivered by inhalation therapy (US Pat. Nos. 5,669,376 and 5,655,516).

As vascular regulators, suitable agents can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration will be by the oral route. The dosage will depend on the age, health and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

As described below, once identified by the methods described herein, there are many ways that such agents could be readily adapted for administration. Determination of the optimal range of effective amounts of each component is within the skill of those in the therapeutic arts and is based on the intended use. In addition to the particular agent selected, the compositions of the present invention include excipients and adjuvants that facilitate processing of the active compound into a formulation that can be used in medicine, for example, for delivery to the site of action. Including a suitable pharmaceutically acceptable carrier,
Other components can be included. Suitable formulations for oral administration include water-soluble variants,
For example, aqueous solutions of the active compounds in water-soluble salts. In addition, suspensions of the active compounds,
And suitably, an oily injection suspension can be administered. Suitable lipophilic solvents or vehicles are fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example,
Contains ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or gintran.

If desired, suspensions can also contain stabilizing agents. Liposomes can also be used to encapsulate a drug for delivery to cells. Pharmaceutical formulations for systemic administration according to the present invention may be formulated for enteral, parenteral or topical administration. fact,
All three types of formulations can be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include soft or hard gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release variants thereof.

Agents of the invention that modulate the sequestration and transport of selected essential membrane and secreted proteins can be provided alone or in combination with another suitable pharmacologically active agent. For example, an agent of the invention that reduces the presence of various cell surface receptors involved in viral envelope attachment can be administered in combination with other anti-viral agents. As used herein, two agents are:
Said two agents are said to be administered simultaneously or in combination if the agents are administered independently so as to act simultaneously.

Example 14: Identification of spectrin or adapter protein domain (s) responsible for binding and transport of essential membrane or secreted proteins. As described in FIG. 2, βIΣspectrin is a specific adapter protein. And / or contains a series of functional domains that have been or have been shown to confer the ability to bind to the protein to be transported. Similarly, ankyrins contain a large number of repeats (13 to 24 repeat units characteristic of most ankyrins), each ankyrin-repeat structure comprising two alpha helices and a β-helix.
Consists of a hairpin loop. The complexity of both individual repeats and the number of repeats in the structure of both structural proteins, such as spectrin, and adapter proteins, such as ankyrin, is due to the large number of distinct essential membrane or secretory proteins in which both molecules are distinguished. To change the ability to bind.

By way of example, in ankyrin, multiple repeating units provide a structure in which the interactions between the helices form a central core structure, while the exposed β-hairpin turn tips provide a putative protein-protein interaction surface. (See FIG. 21). In one scenario, a seven-residue loop in the MAB of α-Na, K-ATPase interacts with a specific site in ankyrin created by the tip of one or more of these β-hairpin turns. Because multiple potential binding pockets will be created by the 13 to 24 repeats characteristic of most ankyrins, specific and unique binding sites are likely to be responsible for ankyrin binding activity in other proteins. May also be present in short peptide sequences.

The ability of an adapter protein to bind spectrin or an essential membrane protein or a secreted protein and bind directly to spectrin is assayed to determine the spectrin domain, or the adapter protein and the essential membrane of a given adapter protein. Domain combinations between proteins or secreted proteins, or responsible for specific membrane protein or secreted protein specific binding, can be identified directly. Similarly, the ability of a given essential membrane or secreted protein to bind to an adapter protein such as ankyrin is assayed to determine the ankyrin domain or combination of domains responsible for specific, essential membrane or secreted protein binding. Can be identified. Thus, the identification of specific domains responsible for binding allows the development of peptide-based molecules that modulate or disrupt the specific binding of many of the protein-protein interactions involved in SAATS.

As an example, to determine the identity of the ankyrin domain responsible for specific binding to a given essential membrane, a series of fragments of the protein were generated using deletion analysis of the ankyrin repeat region. Let it. The fragments are then tested for their ability to bind to the essential membrane protein of interest. Commonly available means of detecting the ability of the ankyrin fragment to bind to the specific essential membrane protein can be used. For example, using a cell-free system where binding is detected by the ability of the ankyrin fragment and detected by a radiolabel, a fluorescent tag, or a specific antibody to be co-immunoprecipitated by an antibody against the protein suspected of binding thereto. I do. Alternatively, the ability of ankyrin fragments to co-precipitate with cells of interest or pre-permeabilized cells or membrane preparations (e.g., co-precipitation of ankyrin with Na, K-ATPase-rich membranes, Morrow et al. , 1989) is another method of detecting binding. Other methods include binding to immobilized ligands; co-precipitation, co-elution on gel filtration chromatography; surface plasmon resonance, fluorescence energy transfer; osmotic pressure; light scattering; resonance Raman spectroscopy, NMR spectroscopy, yeast 2 -Hybrid systems; including expression cloning, and phage display. Other methods will be known to practitioners in the art. Since an individual ankyrin repeat unit is often insoluble or unstable if it is the target of the assay, it is often more avid for binding activity, e.g., from the beta-hairpin loop of the ankyrin repeat structure. It would be worth examining a small internal array. High-throughput assays can also be developed to detect binding of a library of ankyrin fragments containing all possible combinations of repeating units, as well as all individual repeating units. An example of such an assay would be a single, library of beta-hairpin loop sequences of all known assay repeat units into a silo-library suitable for screening in a yeast 2-hybrid or CHO2-hybrid KISS assay for the ligand of interest. Incorporating dimer and trimer combinations (Fear
on et al., 1992). Also, sequences immobilized on nitrocellulose or immobilon or similar membranes, or incorporated into bacteriophage to generate phage display libraries on plastic multiwell plates or suitable for binding to the protein of interest. A similar strategy could be used, utilizing identical peptide sequences, either recombinantly or generated by synthetic methods. (Scott, 1992).

It should be understood that the foregoing discussion and illustrations merely provide a detailed description of certain preferred embodiments. Thus, it will be apparent to one skilled in the art that various modifications and equivalents may be made without departing from the spirit and scope of the invention. All articles, patents and patent applications specified above are hereby incorporated by reference in their entirety.

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[Brief description of the drawings]

FIG. 1. Spectrin / ankyrin / adaptor protein transport / ligation system (SAATS
), Wherein one-way protein transport can be blocked. (A
() Is a general schematic diagram in which dynactin moves vesicles from the endoplasmic reticulum (ER) to the Golgi. Transported tubular vesicle structures / vesicles exiting the exit port of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) bind simultaneously to vesicles and dynactin through interaction with centlactin, which is part of the dynactin complex Can, SAA
Linked to dynactin by TS. Centracin is an ARP that binds actin-related proteins or spectrin. (B) In wild-type cells, the various proteins are bound via adapter proteins such as ankyrin or to another membrane carrier protein which is subsequently bound by SAATS (for secreted proteins). Binds directly or indirectly to vesicle-associated spectrin. "L" represents the lumen of the ER. (C), the selected protein is achieved by blocking its attachment to SAATS, as in cells transfected with the cDNA encoding the spectrin βIN _-5 construct. In such cells, certain essential membrane or secreted proteins are not sequestered in vesicles for transport from the endoplasmic reticulum to the Golgi.

FIG. 2. Suitable SA for blocking membrane protein display and for identifying pharmacological agents
1 shows a series of suitable and representative βIΣ ^* spectrin constructs useful for determining ATS targets. Some of the important functional domains are also depicted. Ankyrin binding occurs at repeating unit 15. The amino-terminal domain (green) confers actin and possibly ARP binding to the spectrin complex. Also shown are the membrane association domains (MADI and MAD2). The third membrane association domain (MAD3) is located somewhere between repeat units 3 and 9. Similar constructs prepared from other spectrins involved in protein transport (eg, βIII spectrin) can also be prepared.

FIG. 3 illustrates the use of cultured Madin Darby canine kidney (MDCK) cells to monitor surface expression of Na, K-ATPase by indirect immunofluorescence and immunoprecipitation. In this example, expression of the amino-terminal βI spectrin construct in MDCK cells disrupts the native Golgi spectrin-ankyrin skeleton and the Na, K-ATPase assembly. (A) The intracellular distribution of wild-type Golgi spectrin was monitored using Mab VIIIC7, which reacts with full-length Golgi spectrin, but not with the shorter FLAG-tagged βIN _-5 construct ( Harris et al., 1985; Devarajan et al., 1996; G
odi et al., 1997). βI in cells transfected with βI _11-C
Σ ^* Spectrin distribution was monitored by Mab VD4, which did not react with the βI _11-C peptide. AnkG119 is an antibody "Jasmin" (Devaraj)
α et al., 1996); α-Na, K-ATPase is
Monitored by Mab from state Biotechnology. Note the dispersion of endogenous Golgi spectrin, AnkG 119 and α-Na, K-ATPase by βI _N-5 (rather than βI _11-C ). All shorter spectrin constructs containing region I and MAD1 exhibit similar effects, while constructs lacking these regions of βI spectrin show βIΣ ^* spectrin, AnkG119, or Na, K-ATPase. Did not significantly affect the natural distribution of Bar = 10 μm. (B), 1 of Na, K-ATPase and spectrin
Interaction with 4-15 repeat units occurs via an ankyrin G119 adapter protein intermediate, as detected by co-precipitation. In wt cells, β
IΣ ^* _Spectrin , Ank _G119 , _Centracin , and Na, K-ATPase are co-precipitated from detergent lysate by Mab VIIIC7 (lane IP)
. Soluble α (detected here by immunoblotting for αII spectrin)
IIβII spectrin, a form predominantly found in the plasma membrane, is also present in these detergent lysates (lanes lys) but is not part of the precipitable Golgi spectrin complex. Control experiments with immature or irrelevant antibodies do not precipitate any of these components (data not shown). Similarly, immunoprecipitation of βI _N-5 transfected cells with Mab VIIIC7 also showed co-precipitation of Ank _G119 and Na, K-ATPase with βIΣ ^* spectrin, which was determined by the βI _N-5 construct. Despite the induced dispersion of these elements, these elements showed to remain associated in the detergent lysate (data not shown). Figure 5 shows a Western blot after SDS-PAGE visualized by ECL.

FIG. 4 is an illustration of another assay demonstrating the impairment of Na, K-ATPase transport from ER induced by SAATS inhibition. Dispersion of Golgi spectrin by βIN _-5 blocks Na, K-ATPase transport to the medial Golgi and its incorporation into the detergent stable scaffold complex. (A), (as measured by Mab VIIIC7 does not react with originating revealed the beta I _N-5 peptide) endogenous Betaaishiguma ^* spectrin in transfected MDCK cells beta I _N-5 spectrin
, And intracellular distribution of Na, K-ATPase. (B), β-Na, K-ATP
Golgi-dependent glycosylation of ase is blocked by βIN _-5 spectrin peptide. Control MDCK cells (wt) or cells transfected with the βIN _-5 construct (βIN _-5 ) were subjected to Western blotting using β-Na, K-ATPase specific antibody (Mab B1-13). Was analyzed by The apparent MW of various products is shown. 50k representing mature glycosylated β-Na, K-ATPase
Broad band exceeding Da (gly) and ER-dependent core β-Na, K-AT
Note the strong band of 44 kDa in βIN _-5 cells (core), which represent Pase glycosylation products. Thus, βIN _-5 spectrin peptide inhibits the process of mature glycosylation of β-Na, K-ATPase and processes characteristic of medial-Golgi processing, but inhibits the formation of core glycosylation products that occur in ER. Does not seem to interfere. In this experiment, almost half of the βIN _-5 cells expressed the βIN _-5 construct, as judged by immunofluorescence (data not shown), which was observed in the βIN _-5 cell line. It appears to account for most of the mature glycosylation products. Band = 106 kDa in wild-type cells ( ^* ) is not uniform in appearance, and in SDCK cells,
As normally observed after S-PAGE (Morrow et al., 1989), β-
It may represent a cross-linking adduct of Na, K-ATPase with α-Na, K-ATPase or other proteins. (C) shows Na present in the soluble and detergent insoluble pools of MDCK cells transfected with different βI spectrin constructs.
, K-ATPase. Fx1 (soluble fraction) was 100 mM Na
Material solubilized by 0.5% Triton X-100 in Cl; Fx2
(Cytoskeletal fraction) are solubilized material by 0.5% Triton X-100 and 250mM NH ₄ S O ₄ (Devarajan et al., 1994). Shown are Western blots for α-Na, K-ATPase after SDS-PAGE. (D) Results of three separate measurements of wt and α-Na, K-ATPase extractability of the two transfected MDCK cell lines were quantified in duplicate by densitometry, and all analyzes were averaged. did. Error bars represent ± ISD. Note the substantial loss of fully assembled and detergent-insoluble Na, K-ATPase in βIN _-5 expressing cells.

FIG. 5 is an illustration of the selective blockade of Na, K-ATPase and VSV-G transport by βIN _-5 but not E-cadherin. 4 shows a comparison of the effects of βIN _-5 spectrin on Golgi and different protein assembly in MDCK cells. Wild type (B, D, F, H) and βIN _-5 transfected cells (A, C, E, G) are shown. Localization of VSV-G protein (E, F) was measured after transient infection. E-cadherin (C, D) is a Mab from the Transduction labs, and a process that occurs in the trans-Golgi, depending on the extent to which the precursor peptide is proteolytically (inserted, western blotted) from 135 kDa ( ^* ) to 120 kDa (EC). ) (Shore and Nelson, 1991). There was no significant difference in the extent of that level of E-cadherin processing or assembly at the plasma membrane in βIN _-5 lines versus wt cells. Na, K-ATPase
Despite disruption of VSV-G transport (and wt Golgi spectrin skeleton, FIG. 3), Golgi is normal by the distribution of β-COP (A, B) and in uranyl acetate and lead stained electron microscopy. (G, H) (arrows), due to the presence of a proximate Golgi structure appearing at the point, appears almost intact. Bar = 10 μ for (A-F), 0.5 μ for (G, H). Original magnification (G, H
) = 63,000 ×.

FIG. 6 is an illustration of the rescue of Na, K-ATPase-transport by including a specific SAATS binding domain that binds to ankyrin. MDCK cells are transformed into the β
Transfected with IN _-5 or βIN _-5,15 . This latter construct incorporates the ankyrin-binding domain of spectrin into the βIN _-5 peptide. In this case, it is measured by its surface display and, secondarily, by a decrease in cell size (resulting from cell swelling due to a lack of plasma membrane Na, K-ATPase in βIN _-5 transfected cells). Also note the complete recovery of Na, K-ATPase transport.

FIG. 7 shows how SAATS (not Na, K-ATPase) VSV-G
It is an explanatory view of whether or not to block. MDCK cells infected with vesicular stomatitis virus were used to monitor VSV-G protein transport. The transport of this protein is usually via Na, K-ATPase (to wt cells and as shown in FIG. 5).
As for the basolateral membrane. This transport is based on Na,
As with K-ATPase (comparison, FIG. 6), it was blocked by SA ATS inhibition with the βIN _-5 construct. However, unlike Na, K-ATPase, VSV-G transport is not restored by the inclusion of the ankyrin binding domain of spectrin (repeats 14-15) in the construct. Thus, Na, KA
VSV-G transport can be selectively blocked without affecting TPase transport.

FIG. 8 is an explanatory diagram for identifying a small peptide sequence that is responsible for the association between Na, K-ATPase and ankyrin. (A) is a schematic diagram showing the five cytoplasmic domains of α-Na, K-ATPase and their relationship to the ankyrin-binding peptide sequence identified here. The codon positions defining each peptide are indicated. Previous studies have established extensive reactivity of ankyrins with cytoplasmic domains II and III and that domain II contributes most of its binding activity (Devarajan et al., 1).
994; Jordan et al., 1995). (B) Each drawn peptide (II-II
C) was prepared as a fusion construct with SjGST and tested at various concentrations for its ability to bind to purified ANK1 (from human red blood cells) or kidney ankyrin (AMK3) from whole MDCK cell lysates. Was. Shown are results from a single experiment. The top panel is Coomassie blue stained SDS-P for each peptide.
AGE analysis, as well as total MDCK cell extracts applied to an affinity column to detect ANK3 binding. Purified erythrocyte ankyrin was used to detect ANK1 binding. Peptide IIA, sequence-SYYQEAKSSK
IMESFKNMVPQQALV- represents the minimum active sequence detected. We refer to this as the minimal ankyrin binding domain (MAB).

FIG. 9 is a diagram illustrating that specific deletion of the SAATS attachment sequence in an essential membrane protein selectively blocks only its transport. In this case, MDCK cells are wild-type Na, K-AT with a FLAG epitope tag added to their NH2-terminus.
Pase, or that codons 142 to 166 have been deleted.
Was transiently transfected with the tagged mutant Na, K-ATPase. These residues correspond to the minimal ankyrin binding domain identified in FIG.
MAB). In both transfections, cells were converted to wt β-Na,
Co-transfection with K-ATPase ensured sufficient β-Na, K-ATPase paired with α-Na, K-ATPase. (Top) FLAG-tagged wt-Na, K-ATPase is usually delivered to the plasma membrane. (Bottom) MAB
FLAG dagged Na, K-ATPase lacking is not assembled at the plasma membrane and is eventually targeted for degradation in the lysosome. In these cells,
Mutant Na, K-ATPase is the only protein whose transport is disrupted.

FIG. 10 is an illustration of selective blockade of Na, K-ATPase transport in normal cells by expression of a small peptide inhibitory drug. Wild-type MDCK cells were transiently transfected with green fluorescent protein linked to a 25 residue MAB peptide. this is,
The deletion is the same sequence that allows the Na, K-ATPase to be retained in the ER and finally lysosomes in FIG. After approximately 1-2 days of culture, cells were fixed and stained for GFP (using anti-GFP antibody (left panel)) or N
a, Stained for K-ATPase (right panel). Expression of GFP alone in MDCK cells (wt) was determined by Na, K-ATPase distribution or (Na, K-A
Did not affect cell size (depending on TPase function). However, GF
When P carries a 25 residue MAB peptide, endogenous Na, K-
ATPase binding was inhibited, so that it was selectively accumulated in the ER and levels on the plasma membrane were reduced (showing two examples labeled "MAB"). As expected, cells containing GFP-MAB also swell significantly. Other protein distributions were not affected in these cells.

FIG. 11 is an illustration of the three-dimensional structure of MAB, which is well suited for the rational design of small molecule drugs with pharmacological actions similar to MAB. This structure is similar to the active GST shown in FIG.
-Measured using carrier-mediated crystallization based on the MAB peptide (Zhang et al., 1997a). FIG. 3 is a triaxial view of the structure of the minimal ankyrin-binding domain of α-Na, K-ATPase. The basic structural motif is that of a seven-residue "loop" on a "stoke" consisting of antiparallel β-strands. (A, C, E): "Loop"
Surface accessibility indication showing the amphipathic surface formed by the method. (B, D, F): Ribbon diagram showing skeleton outline. The seven residue loop is thought to interact with the β-hairpin tip of one or more ankyrin repeat units. Arrows in (C) mark the depth of the indicated structural plane (E and F). It is expected that small molecule homologs that mimic the seven residue loop structure will be effective and selective inhibitors of Na, K-ATPase transport.

FIG. 12 is an illustration of another essential membrane protein of considerable medical interest that links to SAATS via different ankyrin interactions. (A) CFTR (cystic fibrosis transmembrane conductivity regulator) and homologous TNR (transmembrane binding regulatory domain) close to that (T ransmembrane- N ucleotide bind
ing- R egulatory domain) proteins, both dense MD
Present in the soluble (Fx1) and cytoskeletal pool (Fx2) of CK cells. When these cells are surface labeled with biotin, either from the apical surface (lane A) or the basolateral surface (lane B), both CFTR and TNR are highly polarized,
It is also clear that, unlike Na, K-ATPase, it is predominantly expressed in the apical domain of these polarized epithelial cells. (B) Despite the fact that CFTR and TNR are tip proteins, they nevertheless bind to components of the SAATS system that are responsible for their transport. Whole cell lysates (L) of cells extracted with RIPA buffer, or Fx1 and Fx2, are irrelevant antibody controls (C)
Or with an anti-CFTR antibody (IP). The washed immunoprecipitates were then analyzed by SDS-PAGE and any ankyrins were examined by Western blotting. In exactly the same way that Na, K-ATPase binds to SAATS, both _ankyrins (Ank _G119 and AnkR) are CF
Co-immunoprecipitated with TR. However, CFTR and Na, K-ATPase
Bind at distinct sites on SAATS, allowing their selective modulation.

FIG. 13 shows integrin display by SAATS in cultured endothelial cells.
FIG. 4 is a diagram for explaining the up and down regulation. SV40 transformed mice
Β endothelial cells_N-5Or βI_N-5,15And the surface display of three different integrins was measured by flow cytometry. βI _N-5 Note the blockade of alphaV, beta3, and beta1 integrins by the peptide. Conversely, βI_N-5,15The peptides were alpha V and beta 1
Significantly enhances Glin's surface display (beta 3 integrin does not)
No). This augmented display (as opposed to blocking) is probably
Select a Peptide or Agent that Enhances Binding of a Membrane Protein to SAATS
This enhances the efficiency of SAATS-directed luggage loading for vesicle transport.
Achieved.

FIG. 14 is an illustration of an additional example of SAATS regulation of integrin display in cultured endothelial cells. The experiment was performed as in FIG. Alpha-V
Note that integrins are modulated by SAATS. Also note that in this example, βIN _-2,15 was used to rescue beta-1 expression. Although βI _N-2 is an inhibitor that more widely blocks SAATS transport (see FIG. 1), in this example, extraordinary levels of beta-1 result from inclusion of repeats 14,15 into the βI _N-2 peptide. Can be achieved.

FIG. 15 is an illustration of PECAM (CD31) surface modulation by SAATS inhibitory peptides using two different epitope tags, FLAG and GFP. Cells were transfected as described above and surface display of PECAM, an intercellular adhesion molecule of the IgG superfamily, was monitored by flow cytometry. Note that for both constructs, inclusion of the βIN _-5 sequence indicates strong blockade of transport, indicating involvement with SAATS. In both cases, the surface display was rescued or complemented by the βIN _-5,15 peptide. This effect is likely due to the interaction of GFP with the COOH-terminal 15th repeat unit required to restore SAATS binding activity to PECAM, with the N-terminal FLAG-tagged construct versus the COOH-terminal GFP dugd construct. It was somewhat more pronounced.

FIG. 16 illustrates the modulation of CD45 and TNFR-1 in T-lymphocytes by SAATS inhibitors. In this experiment, Jurkat T-lymphocytes were transfected with the indicated construct, including βI _{14 -15} alone. By the construct lacking repeat 15 as well as [lacking the constitutive Golgi targeting signal (see Figure 2)]
Note the marked down-regulation of the described ankyrin-binding protein, CD45, by the _I14-15 peptide itself. These experiments are based on T
NFR-1 display is (rather than blocked) by βIN _-2 or βIN _-5
Rather, it is also illustrated to be up-regulated, suggesting that, like E-cadherin, its attachment to SAATS is mediated directly or indirectly by sequences contained within βIN _-2 . I do.

FIG. 17 is a diagram illustrating the modulation of Fas and Fas-L in T-lymphocytes by SAATS. The experiment was as described above. Note the changes in both Fas and Fas-L due to βIN _-5 and βIN _-5,15 peptides.

FIG. 18 is an illustration of the use of antibodies or small peptides injected that modulate Na, K-ATPase in wild-type MDCK cells. In the cluster of wild type MDCK (A
) GST alone; or (B) β produced as a recombinant fusion peptide with GST.
The _IN-4 peptide was microinjected and then stained for Na, K-ATPase by indirect immunofluorescence. Microinjected G
Na, K-A in the injected cells on the left side of (B), while ST had no effect.
Note the loss of TPase staining intensity. Impaired Na, K-ATP to membrane
The results of ase delivery are also evident in the larger size of cells with insufficient Na, K-ATPase, as well as the changes observed when βIN _-5 is transfected. These results indicate that selective blockade of Na, K-ATPase can be achieved by exogenous small peptides. Similar experiments were performed using various monoclonal antibodies. In these experiments, cells were in confluence and all cells in the isolated cluster were injected. (C) Control injection of buffer alone has no effect on the distribution of Na, K-ATPase. (D) (SAAT
Microinjection of Mab II D2 that exclusively reacts with αI spectrin has no effect on Na, K-ATPase. (E) Ma reacting with βI spectrin (Harris et al., 1986)
b Microinjection of VIIIC7 leads to severe disruption of Na, K-ATPase delivery, considerable accumulation within the cells, and swelling of the cells. In this micrograph, the loss of the membrane Na, K-ATPase is too severe and the boundaries of the membrane cannot be identified. This experiment demonstrates the effect of a SAATS monoclonal antibody in blocking SAATS activity.

FIG. 19 presents the full length cDNA sequence of a novel isoform of βIII spectrin, which may be an additional component of SAATS. (A) The partial cDNA clone identified as the expressed sequence (EST) was identified as a member of the spectrin family, extended to completion by 5'RACE PCR amplification and sequenced. (B) The resulting full-length sequence is predicted to encode a novel isoform of spectrin called βIII (Morrow, 1997), similar to βI and βII, but shows significant differences in selected regions . (C) shows sequence similarity between various beta-spectrins. Also, β against βI and βII spectrin
A phylogenetic diagram showing the approximate relationship of III spectrin is also shown.

FIG. 20 illustrates examples of SAATS inhibitory peptide constructs in two different vectors: pcDNA3 (FLAG-tagged) and enhanced green fluorescent protein (eGFP).

FIG. 4B shows a model of how Na, K-ATPase can interact with one or more ankyrin repeat units.

FIG. 22. Cystic fibrosis transmembrane conductance regulator (CFT) by SAATS
R) shows the modulation of the surface display. MDCK cells were transfected with beta I _N-5 or beta I _{N-5,1 5} construct, then surface labeled with biotin. After labeling, cells were solubilized and all CFTR in each culture was immunoprecipitated with anti-CFTR antibody. The precipitate is then analyzed by SDS-PAGE, transferred to nitrocellulose,
Surface CFTR was detected by avidin labeling. Total CFTR was also measured in these culture extracts by Western blotting (data not shown). In all cell lines, there was minimal change in total CFTR in the cells. However, there was a marked reduction in the surface display of CFTR induced by the SAATS blocker βIN _-5 , while the βIN _-5,15 construct significantly enhanced the surface display of CFTR. Taken together, these results demonstrate the involvement of SAATS in CFTR delivery, and a possible treatment for the control of diseases such as cystic fibrosis, or related proteins such as the multidrug resistance gene product (MDR). Suggest a strategic approach.

FIG. 23 is a schematic diagram of a spectrin heterodimer.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12N 15/00 A 5/10 A61K 37/02 G01N 33/53 C12N 5/00 B F term (Ref.)

Claims (27)

[Claims] 1. For transport from the endoplasmic reticulum to the cis-Golgi apparatus, or from the cis-Golgi to the medial-Golgi apparatus, or from the medial-Golgi apparatus to the trans-Golgi apparatus, or from the trans-Golgi apparatus to the plasma membrane. A method for modulating the intracellular sequestration of a selected essential membrane protein or secreted protein into a transport vesicle, wherein the method is effective to enhance or inhibit the binding of the selected protein to the transport vesicle Then contacting the cells with the compound, thereby enhancing protein sequestration if binding is enhanced or, if binding is inhibited, protein sequestration is inhibited The method comprising: 2. The binding and sequestration of a selected protein is inhibited, wherein the compound comprises: i) a modified or truncated scaffold component of a transport vesicle; ii)
2. The method of claim 1, wherein the method is selected from the group consisting of an associated adapter protein, or iii) a small molecule mimetic of either 1) or ii). 3. The sequestration of a selected protein is inhibited, wherein the compound is a modified or truncated adapter protein, or a small molecule mimetic thereof, wherein the protein or mimetic is a selected protein. 2. The method of claim 1, which mediates the binding or association of the vesicles to the skeletal components of the transport vesicle. 4. The method according to claim 3, wherein the adapter protein is ankyrin and the backbone component of the transport vesicle is spectin. 5. The method of claim 4, wherein said ankyrin is AnkG119. 6. The backbone component or associated adapter protein is ADP-ribosylation factor (AFR), ankyrin, AnkG119 spectin, spectin βI, spectin βIΣ ^* , centlactin and tubulin, and any of the foregoing small molecule mimetics. The method according to claim 2 or 3, wherein the method is selected from the group consisting of: 7. The method according to any one of claims 1 to 5, wherein transport of the selected protein from the endoplasmic reticulum to the cis-Golgi apparatus is inhibited. 8. The method of claim 1, wherein the candidate compound is from the endoplasmic reticulum to the cis-Golgi apparatus, from the cis-Golgi apparatus to the medial-Golgi apparatus, or from the medial-Golgi apparatus to the trans-Golgi apparatus, or from the trans-Golgi apparatus. A method for determining whether to inhibit or enhance the transport of an essential membrane protein or a secreted protein selected via a transport vesicle to a cell membrane, comprising the steps of: And then determining whether vesicular trafficking of the selected protein is enhanced or inhibited. 9. The determination that inhibition of intracellular transport has occurred i) detecting a substantial decrease in the presence of the selected protein in the cell membrane or in the extracellular medium; or ii) to the Golgi apparatus and to the Golgi. 9. The method of claim 8, wherein the method is performed by detecting a dispersed intracellular presence of the selected protein outside of its normal pattern of transport through the device. 10. The vesicle transport of selected essential membrane or secretory proteins from the endoplasmic reticulum to and through the Golgi apparatus, the transport of the selected protein to the components of the endoplasmic reticulum backbone. Determining whether mediated by binding, treating cells expressing the selected protein with an agent that interferes with sequestration of the essential membrane or secreted protein into transport vesicles; Then determining whether the selected protein is substantially found in the cell in its normal distribution pattern, whereby the abnormal distribution pattern indicates mediated vesicle transport. Method. 11. The method according to claim 11, wherein the agent is one of a series of progressively more truncated portions of a protein that is a structural component of the vesicle skeleton or that is an associated adapter protein. 11. The method of claim 10, wherein the sequence has been separately transfected with cDNA encoding the vesicle backbone or associated adapter protein. 12. The method according to claim 12, wherein said vesicle skeleton or associated adapter protein is AD.
The method according to claim 11, wherein the method is selected from the group consisting of P-ribosylation factor (ARF), ankyrin, AnkG119 spectrin, spectrin βI, spectrin βIΣ ^* , centlatin and tubulin. 13. The step of determining whether the distribution pattern of the selected protein is substantially normal comprises: 1) substantially reducing the presence of the selected protein in its normal subcellular distribution pattern; or ii. 2.) assessing whether there is a decrease in the presence of the selected protein outside the cell membrane or in the extracellular medium of the cell.
The method according to 0. 14. The vesicle backbone protein is spectrin.
The method described in. 15. The spectrin is βIΣ ^* spectrin.
The method described in. 16. The adapter protein of claim 12, wherein the adapter protein is ankyrin.
The method described in. 17. The method of claim 16, wherein said ankyrin is AnkG119. 18. A group of βIΣ ^* spectrin cDNA constructs of various lengths each encoding one of a series of complementary and overlapping truncated βIΣ ^* spectrin peptides. 19. A set of individual clonal cell lines each expressing one of the βIΣ ^* spectrin cDNA constructs of claim 18. 20. An isolated, purified nucleic acid fragment encoding βIII spectrin. 21. The nucleic acid of claim 20, wherein said βIII spectrin comprises the amino acid sequence of FIG. 19B. 22. The nucleic acid of claim 20, comprising the sequence of FIG. 19A. 23. An isolated nucleic acid that specifically hybridizes under stringent conditions to the nucleic acid of claim 22. A vector comprising the nucleic acid according to any one of claims 20 to 23. 25. A host cell comprising the vector according to claim 24. 26. For transport from the endoplasmic reticulum to the cis-Golgi apparatus, or from the cis-Golgi apparatus to the medial-Golgi apparatus, or from the medial-Golgi apparatus to the trans-Golgi apparatus, or from the trans-Golgi apparatus to the cell membrane. A relatively excess of a particular essential membrane protein, characterized by administering a unit dose of the compound effective to inhibit the intracellular sequestration of the selected essential or secreted protein into the transport vesicle How to reduce the effects of the disease caused. 27. Transport vesicles from the endoplasmic reticulum to the cis-Golgi apparatus, or from the cis-Golgi apparatus to the medial-Golgi apparatus, or from the medial-Golgi apparatus to the trans-Golgi apparatus, or from the trans-Golgi apparatus to the cell membrane. By administering a unit dose of a compound effective to enhance the intracellular sequestration of a selected essential or secreted protein into its transport, by a relative lack of an amount of a particular essential membrane protein How to reduce the effects of the disease caused.