COMPOSITIONS AND METHODS FOR TREATING CANCER

STATEMENT OF GOVERNMENT SUPPORT

[001] This invention was made with government support under GM064387 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.

BACKGROUND

[002] Clathrin is normally involved in receptor-mediated endocytosis (RME), but its function can be redirected to promote cancer suppressor p53-mediated transactivation. To do this, trimeric clathrin must be made monomeric, but how this occurs is not understood. The characteristic shape of clathrin (Figure 1 ) enables this three-legged molecule to self-assemble into polyhedral coats in receptor- mediated endocytosis (RME). Although the formation of clathrin lattices is energetically favored and spontaneous, RME requires a host of accessory proteins. Adaptor proteins [1 , 2, 3], non-visual arrestins [4, 5], and Hsc70 [6, 7, 8, 9] associate with the N-terminal β-propeller domain of clathrin heavy chain (CHC) [10, 1 1 ]. This propeller domain is joined to a filamentous portion (aa543-1576) that is subdivided into the distal and proximal domains with a flexible knee joint between them [12, 13] and ends in the trimerization domain [14, 15]. The proximal domain in each leg binds two types of clathrin light chain subunits (LCa and LCb [16, 17, 18]) in mammals and one light chain in yeast clathrin [19]. Functionally, light chain associates with Huntingtin-lnteracting Proteins (HIPs) [20, 21 , 22], is thought to modify the flexibility of the knee joint to regulate lattice formation [13], and contributes to the stability of the trimerization domain [23, 24, 25].

[003] There is a body of evidence that shows clathrin is not limited to

endocytosis [26, 27, 28, 29]. Most surprising is the finding that clathrin has a role in transactivation of the cancer suppressor p53 in the nucleus [30, 31 , 32]. In this capacity, trimeric clathrin must be made monomeric [31 ], but the details of how this happens are not known.

SUMMARY

[004] In a first aspect, there is provided a modified clathrin heavy chain polypeptide, or an active fragment thereof, comprising an amino acid replacement of cysteine with an amino acid other than cysteine in an unmodified clathrin heavy chain polypeptide at a locus corresponding to amino acid cysteine-1573 of a clathrin heavy chain polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1 .

[005] In a second aspect, there is provided a modified clathrin heavy chain polypeptide comprising the amino acid sequence of Formula (1 ):

(1 -1572)-X (1574-1675) Formula (1 )

[006] In Formula (1 ), Xi is an amino acid residue selected from the group consisting of Ala, Val, Leu, lie, Met, Gin, Glu, Gly, His, Met, Ser, Thr, Trp, and Tyr; (1 -1572) is a peptide chain including the first 1572 amino acid residues of the human CHC polypeptide counted from the N-terminal end or an analog thereof or derivative thereof. (1574-1675) is a peptide chain including amino acid residues 1574 to 1675 of the human CHC polypeptide, or an analog thereof or a derivative thereof.

[007] Other objects and features will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE FIGURES

[008] Figure 1 illustrates the clathrin trimerization domain structure without light chains.

[009] Figure 2 illustrates the identification of a topological switch in clathrin.

[0010] Figure 3 illustrates nuclear localization of clathrin hub following specific mutations in helix 7j.

DEFINITIONS

[0011] When introducing elements of aspects of the invention or the

embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "containing," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. The term "or" means any one member of a particular list and also includes any combination of members of that list, unless otherwise specified.

[0012] As used herein, a "clathrin heavy chain" polypeptide (also referred to herein as CHC) refers to any CHC polypeptide (protein) including, but not limited to, recombinantly-produced polypeptide, synthetically-produced polypeptide and CHC extracted from cells. Clathrin heavy chain polypeptides include precursor clathrin heavy chain polypeptide having signal sequences and mature clathrin heavy chain polypeptides. CHC polypeptides include related polypeptides from different species including, but not limited to animals of human and nonhuman origin. CHC polypeptide amino acid sequences can contain varying number of amino acid residues. For example, the human CHC of SEQ ID NO: 1 polypeptide has 1675 amino acids. Other polypeptides also can be shorter than 1675 amino acids, provided that a polypeptide retains an activity of the clathrin heavy chain. The clathrin triskelion is composed of three clathrin heavy chains and three light chains interacting at their C-termini. The three heavy chains provide the structural backbone of the clathrin lattice, and the three light chains are thought to regulate the formation and disassembly of a clathrin lattice. Human CHC's include allelic variant isoforms among individuals, alternative splice variants, synthetic molecules from nucleic acids, protein isolated from human tissue and cells, and modified forms thereof.

[0013] CHC polypeptides exhibit allelic variation and species variation. Included, in various aspects, are fragments of CHC that are of sufficient length to be functionally active; such fragments are known and/or can be identified by assays to determine the promotion of p53-mediated transactivation. CHC polypeptides have been isolated from a variety of species. Exemplary CHC polypeptides of non- human origin include, but are not limited to, bovine, murine, amphibian, insect, and avian CHC polypeptides, and include, but are not limited to CHC, allelic variant isoforms, synthetic molecules produced from encoding nucleic acid molecules, protein isolated from non-human tissue and cells, and modified forms thereof. Non- human CHC also includes fragments of CHC that are of sufficient length to be functionally active.

[0014] As used herein, an "activity" or "property" of a CHC polypeptide (protein) refers to any activity or property exhibited by a CHC protein that can be assessed. Such activities include those observed or exhibited in vitro or in vivo (typically referred to as a biological activity). These activities include, but are not limited to, nuclear localization and the promotion of p53-mediated transactivation.

[0015] As used herein, a "fragment of CHC polypeptide" refers to any fragment that exhibits one or more biological activities of the full-length polypeptide.

[0016] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids), as well as other modifications known in the art.

[0017] As used herein, "unmodified polypeptide," "unmodified protein,"

"unmodified clathrin," "unmodified CHC polypeptide," or grammatical variations thereof, refer to a starting polypeptide (protein) that is selected for modification. The starting unmodified target polypeptide can be the naturally occurring, wild type (WT) form of a protein. In addition, the starting unmodified polypeptides previously can have been altered or mutated, such that they differ from the native wild-type isoform, but are nonetheless referred to herein as starting unmodified polypeptides relative to the subsequently modified polypeptides disclosed herein. Thus, existing polypeptides known in the art that have previously been modified to have a desired increase or decrease in a particular activity compared to an unmodified reference protein can be selected and used herein as the starting "unmodified protein." For example, a polypeptide that has been modified from its native form by one or more single amino acid changes and possesses either an increase or decrease in a desired activity, such as anti-cancer therapeutic activity, can be utilized with the methods provided herein as the starting unmodified polypeptide for further modification of either the same or a different activity.

[0018] Likewise, existing polypeptides known in the art that previously have been modified to have a desired alteration, such as an increase or decrease, in a particular activity compared to an unmodified or reference protein can be selected and used as provided herein for identification of structurally homologous loci on other structurally homologous polypeptides. For example, a polypeptide that has been modified by one or more single amino acid changes and possesses either an increase or decrease in a desired activity (e.g., localization in the nucleus) can be utilized with the methods provided herein to identify structurally homologous polypeptides, corresponding structurally homologous loci that can be replaced with suitable replacing amino acids and tested for either an increase or decrease in a desired or selected activity.

[0019] As intended herein, "modified polypeptide," "modified protein," "modified clathrin," "modified CHC polypeptide," "derivative," or grammatical variations thereof, refer to derivatives of a protein which may be obtained, for example, by subjecting an unmodified protein, for example wild-type CHC, to one or more modifications. Example modifications include mutations, truncations, enzymatic digestions, and/or changing its post-translational modifications. Mutations may be one or more amino acid replacements, insertions, deletions and/ or any combination thereof.

[0020] As used herein, "in a position or positions corresponding to an amino acid position" of a protein refers to amino acid positions that are determined to correspond to one another based on sequence and/or structural alignments with a specified reference protein. For example, a position corresponding to an amino acid position of human CHC set forth as SEQ ID NO: 1 can be determined empirically by aligning the sequence of amino acids set forth in SEQ ID NO: 1 with a particular polypeptide of interest. Corresponding positions can be determined by such alignment by one of skill in the art using manual alignments or by using the numerous alignment programs available (for example, BLASTP). Corresponding positions also can be based on structural alignments, for example, by using computer simulated alignments of protein structure. Recitation that amino acids of a polypeptide correspond to amino acids in a disclosed sequence refers to amino acids identified upon alignment of the polypeptide with the disclosed sequence to maximize identity or homology (where conserved amino acids are aligned) using a standard alignment algorithm, such as the GAP algorithm. As used herein, "at a position corresponding to" refers to a position of interest (e.g., base number or residue number) in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein. The position of interest to the position in another reference protein can be in, for example, a precursor protein, an allelic variant, a heterologous protein, an amino acid sequence from the same protein of another species, and the like. Corresponding positions can be

determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues. For example, identity between the sequences can be greater than 95%, greater than 96%, greater than 97%, greater than 98% and more particularly greater than 99%. The position of interest is then given the number assigned in the reference nucleic acid molecule or polypeptide sequence. One of skill in the art would understand that for a modified CHC polypeptide compared to an unmodified CHC polypeptide, amino acid residue 1 of the modified polypeptide corresponds to amino acid residue 1 of the unmodified CHC

polypeptide. One of skill in the art would also understand that for a modified precursor CHC polypeptide compared to a precursor unmodified CHC polypeptide, amino acid residue 1 of the modified polypeptide corresponds to amino acid residue 1 of the unmodified CHC polypeptide.

[0021] As used herein, the term "nucleic acid molecule" encompasses both deoxyribonucleotides and ribonucleotides and refers to a polymeric form of nucleotides including two or more nucleotide monomers. The nucleotides can be naturally occurring, artificial (such as PNA and XNA), modified, and unusual nucleotides such as those referred to in 37 C.F.R. §§1 .821 -1 .822. Examples of nucleic acid molecules include oligonucleotides that typically range in length from 2 nucleotides to about 100 nucleotides, and polynucleotides, which typically have a length greater than about 100 nucleotides.

[0022] As used herein, the terms "homology" and "identity" are used

interchangeably but homology for proteins can include conservative amino acid changes. Usually, to identify corresponding positions the sequences of amino acids are aligned so that the highest order match is obtained (see, for example,:

Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griflin, A. M., and Griflin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and

Sequence Analysis Primer, Gribskov, M. and Devereux, J ., eds., M Stockton Press, New York, 1991 ; Carillo et al. SIAM.I. Applied Math 48: 1073 (1988)).

[0023] As use herein, "sequence identity" refers to the number of identical amino acids (homology includes conservative amino acid substitutions as well). Sequence identity can be determined by standard alignment algorithm programs, and used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid or along at least about 70%, 80% or 90% of the full length nucleic acid molecule of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule. (For proteins, for determination of homology conservative amino acids can be aligned as well as identical amino acids; in this case percentage of identity and percentage homology vary). Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" can be determined using known computer algorithms such as the "FAST A "program," using for example, the default parameters as in Pearson et al. Proc. Natl. Acad. Sci. USA 85: 2444 (1988) (other programs include the GCG program package (Devereux, J ., et al., Nucleic Acids Research 12(1 ): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., .1 . Molec. Biol. 215: 403 (1990); Guide to Huge Computers, Martin J . Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. SIAM. I. Applied Math 48: 1073 (1988)). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar" "MegAlign" program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) "Gap" program (Madison Wis.)). Percent homology or identity of proteins and/ or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g., Needleman et al. .1 . Mol. Biol. 48: 443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2: 482 (1981 )). Briefly, a GAP program defines similarity as the number of aligned symbols (e.g., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1 ) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) and the weighted comparison matrix of Gribskov et al. Nucl. Acids Res. 14: 6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3 .0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Therefore, as used herein, the term "identity" represents a comparison between a test and a reference polypeptide or polynucleotide.

[0024] As used herein, the term "at least 90% identical to" refers to percent identities from 90 to 100% relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) of amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. , 10/100 amino acid difference (approximately 90% identity).

Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.

[0025] As used herein, an "amino acid replacement" refers to the replacement of one amino acid by another amino acid. The replacement can be by a natural amino acid or non-natural amino acids. When one amino acid is replaced by another amino acid in a protein, the total number of amino acids in the protein is unchanged.

[0026] As used herein, the phrase "pseudo-wild type," in the context of single or multiple amino acid replacements, are those amino acids that, while different from the original, such as native, amino acid at a given amino acid position, can replace the native one at that position without introducing any measurable change in a particular protein activity. A population of sets of nucleic acid molecules encoding a collection of mutant molecules is generated and phenotypically characterized such that proteins with sequences of amino acids different from the original amino acid, but that still elicit substantially the same level (i.e., at least 10%, 50%, 70%, 90%, 95%, 100%, depending upon the protein) and type of desired activity as the original protein are selected.

[0027] As used herein, "a naked polypeptide chain" refers to a polypeptide that is not post-translationally modified or otherwise chemically modified, and only contains covalently linked amino acids. [0028] As used herein, a "polypeptide complex" includes polypeptides produced by chemical modification or post-translational modification. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, famysylation, phosphorylation and/or other polypeptide modifications known in the art.

[0029] As used herein, the amino acids, which occur in the various sequences of amino acids provided herein, are identified according to their known, three-letter or one-letter abbreviations.

[0030] As used herein, "naturally-occurring" amino acids refer to the 20 L-amino acids that occur in polypeptides. As used herein, the term "non-natural amino acid" refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids, thus, include amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids.

[0031] As used herein, an amino acid is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids non-natural amino acids, and amino acid analogs.

[0032] As used herein, an amino acid residue is an amino acid molecule that has lost a hydrogen atom or a hydroxyl moiety by becoming joined to another amino acid molecule. When joined to two other molecules of amino acid(s), the residue has lost both a hydrogen atom and a hydroxyl moiety, thereby having lost a water molecule.

[0033] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: lessening severity, alleviation, and/or removal of one or more symptoms associated with cancer. Treatment also encompasses any pharmaceutical use of the modified CHC's and compositions provided herein.

[0034] An "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to affect beneficial or desired results including clinical results, for example in the treatment of cancer. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to treat, ameliorate, reduce the intensity of and prevent cancer. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved when administered in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0035] An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs and horses), primates, mice and rats.

[0036] As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.

Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline. Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The

Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

DETAILED DESCRIPTION

[0037] The present invention is based on the discovery that localization of clathrin in the nucleus is preceded by its detrimerization in the cytosol, and that detrimerization is dependent on a topology switch near the center of the clathrin trimerization domain of the clathrin heavy chain. This switch, as illustrated in Figure 2, includes helix 7j, helix Tx1 and a segment, labeled pCS, which is predicted to undergo a conformational change. It was also found that a single cysteine in helix 7j, specifically cysteine-1573 in the Bos taurus clathrin having NCBI Reference Sequence Number NP_776448, is crucial for clathrin detrimerization and is key to the ability of clathrin hub to enter the nucleus. This helix 7j cysteine is also numbered cysteine-1573 in the Homo sapiens clathrin heavy chain (CHC) having NCBI Reference Sequence Number NP_004850.1 (SEQ ID N0:1 ), a sequence having the same heavy chain numbering scheme as NP_776448 (SEQ ID N0:2) (the numbering scheme from NP_776448 is used throughout, unless otherwise specified).

[0038] In all cell types tested, the nuclear localization of the clathrin hub significantly increased when cysteine-1573 was changed to alanine, as compared to wild-type (WT) clathrin. This is significant because the activation of the p53 pathway to suppress cancer formation is linked to the localization of monomeric clathrin in the nucleus. It was also found that a histidine in the topology switch, numbered histidine-1589, is important to clathrin trimerization, although apparently not as important as cysteine-1573.

[0039] The above discoveries are based on the X-ray structure of a clathrin minimum trimerization domain (CTXD, aa1521 -1654). These sub-fragments associate into a native-like trimer, but there are differences when compared to assembled clathrin or clathrin hub. Helix 7 is offset by approximately 10 A and a short helix is unfolded, suggesting they are components of a topology switch that controls trimeric clathrin stability. To test this topology switch hypothesis, residues cysteine-1573 in helix 7j and histidine-1589, just before the destabilized short helix labeled pCS, were mutated in Bos taurus clathrin hub (aa1074-1675) and assessed by confocal microscopy. The microscopy results show HA-tagged WT hub is almost exclusively cytosolic/membrane in non-cancer and cancer cells, but HA-tagged C1573A hub is also localized to the nucleus. The H1589C mutant has an intermediate effect in cancer cells. Without being bound to any particular theory, it is believed that these data support the hypothesis that cysteine-1573 in helix 7j of the topology switch plays an important role in detrimerizing clathrin in the cytosol, and is therefore important in the function of monomeric clathrin in p53-mediated cancer suppression.

Modified Clathrins

[0040] Based on the above discoveries, there are provided modified clathrins that are characterized by a higher tendency to detrimerize and localize to the nucleus than naturally-occurring, wild-type (WT) clathrin. Given a sample of cells featuring one such modified clathrin, modified clathrins may be grouped into three categories, as measured by the number of cells in the sample exhibiting nuclear localization of a given modified clathrin: minimally localizing, having at least 25% but less than 50% of the cells exhibiting nuclear localization; moderately localizing, where at least 50% but less than 75% of the cells exhibit nuclear localization; and highly localizing, where at least 75% of the cells exhibit nuclear localization. It must be borne in mind, however, that the tendency of a clathrin derivative to concentrate in the nucleus is not entirely dependent on the derivative itself, as the degree of localization may also vary somewhat depending on other factors such as the nature of the cells it is measured in (see, for example, Figure 3).

[0041] The modified clathrins are impaired in their ability to form trimers, for example as a result of amino acid replacement by means of mutagenesis. This can be achieved by subjecting clathrin to mutations in the topology switch illustrated in Figure 2, thereby changing one or more of the amino acids in the switch.

Exemplary among such mutants are those bearing mutations in helix 7j, in particular those where the cysteine residue labeled cysteine-1573 (Cys-1573) has been mutated to another amino acid, and in particular to an uncharged or hydrophobic amino acid. Also included are mutants where histidine-1589 (His-1589) is mutated to another amino acid.

[0042] Accordingly, there are provided nucleus-localizing, modified CHC polypeptides including an amino acid sequence obtained by deletion, replacement, addition, or insertion of at least one amino acid residue of the topology switch of unmodified CHC polypeptide. The modified CHC polypeptides provided herein include precursor forms and mature forms; modifications are described with respect to the mature form, but also include modified precursor polypeptides.

Corresponding positions on a particular polypeptide may be determined, for example, by alignment of unchanged residues.

[0043] Exemplary modified CHC polypeptides provided herein contain an amino acid replacement at the locus corresponding to amino acid position 1573 of human clathrin heavy chain compared to unmodified human clathrin heavy chain, where a human clathrin heavy chain comprises a sequence of amino acid residues as set forth in SEQ ID NO:1 . Such modified polypeptides include proteins comprising the sequence of SEQ. ID. NO:1 where the Cys residue at the 1573^th position is replaced with any other amino acid residue except cysteine. In some representative instances, the amino acid residue replacing said Cys is selected from the group consisting of Ala, Val, Leu, lie, Met, Gin, Glu, Gly, His, Met, Ser, Thr, Trp, and Tyr. An uncharged or hydrophobic residue, such as Ala, Val, Leu, He, or Met, is preferred. For example, in polypeptides comprising the sequence of SEQ ID. NO:3, said Cys residue is replaced with an Ala residue.

[0044] Optionally, the His residue at the locus corresponding to the 1589^th position of the amino acid sequence of SEQ ID NO:1 is replaced with any other amino acid residue except histidine. Preferably, the His residue at the 1589^th position is replaced with an uncharged or hydrophobic residue, such as Ala, Val, Leu, lie, or Met. In polypeptides comprising the amino acid sequence of SEQ ID NO:4, for instance, said His residue is replaced with an Ala residue.

[0045] In some examples, the modified CHC polypeptides comprise an amino acid sequence of Formula (1 ):

(1 -1572)-X (1574-1675) Formula (1 ) wherein

Xi is an amino acid residue selected from the group consisting of Ala, Val, Leu, lie, Met, Gin, Glu, Gly, His, Met, Ser, Thr, Trp, and Tyr, (1 -1572) is a peptide chain including the first 1572 amino acid residues of the human CHC polypeptide counted from the N-terminal end or an analog thereof or derivative thereof, and (1574-1675) is a peptide chain including amino acid residues 1574 to 1675 of the human CHC, or an analog thereof or a derivative thereof. In preferred examples, Xi is selected from the group consisting of Ala, Val, Leu, lie, and Met. It is to be understood that there are allelic variants, species variants and isoforms of the polypeptide whose sequence is set forth in SEQ ID NO: 1 , such polypeptides also can be modified at loci corresponding to those of the polypeptides exemplified herein.

[0046] Further examples of the modified CHC polypeptides comprise the amino acid sequence of Formula (2):

(1 -1572)-X (1574-1588)-X₂-(1590-1675) Formula (2) wherein

Xi is an amino acid residue selected from the group consisting of Ala, Val, Leu, lie, Met, Gin, Glu, Gly, His, Met, Ser, Thr, Trp, and Tyr, (1 -1572) is a peptide chain including the first 1572 amino acid residues of human CHC counted from the N- terminal end or an analog thereof or derivative thereof, (1574-1588) is a peptide chain including amino acids 1574 to 1588 of human CHC or an analog thereof or a derivative thereof. X₂ is an amino acid residue selected from the group consisting of Ala, Val, Leu, lie, Met, Gin, Glu, Gly, Cys, Met, Ser, Thr, Trp, and Tyr, and (1590- 1675) is a peptide chain consisting of amino acids 1590 to 1675 of human CHC or an analog thereof or a derivative thereof. In some instances, Xi and X₂ are each independently selected from the group consisting of Ala, Val, Leu, lie, and Met.

[0047] In some cases, the modified CHC polypeptide is a naked polypeptide chain. In other cases, the polypeptide is a polypeptide complex further comprising post-translational modifications, such as glycosylated residues. The polypeptide may include other modifications known in the art. Hence, also provided are modified CHC polypeptide where the CHC is pegylated, albuminated, glycosylated, or has undergone other modifications. Also provided are modified CHC

polypeptides further having one or more pseudo-wild-type mutations.

Representative pseudo-wild-type mutations include deletion, replacement, addition, insertion or a combination thereof of the amino acid residue(s) of an unmodified CHC.

Nucleic Acid Molecules and Vectors

[0048] In another aspect, there are provided nucleic acid molecules comprising polynucleotide sequences which code for a modified CHC polypeptide as described herein. Also provided are vectors comprising such polynucleotide sequences and host cells containing such nucleic acid molecules or vectors. The modified CHC polypeptide may be produced by expressing a polynucleotide sequence encoding the modified CHC polypeptide in a suitable host cell by standard techniques. The modified CHC polypeptide is either expressed directly or as a precursor molecule which has an N-terminal or C-terminal extension, such as a His-tag. Polynucleotide sequences coding for the modified CHC polypeptide may be prepared synthetically by established standard methods, e.g. the phosphoramidite method with an automatic DNA synthesizer and/or by polymerase chain reaction (PCR).

[0049] In a further aspect, there is provided a vector which is capable of replicating in a selected microorganism or host cell and which carries a

polynucleotide sequence encoding a modified CHC as described herein. The recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini- chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. The vector may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. In one representative instance, the recombinant expression vector is capable of replicating in yeast. Examples of sequences which enable the vector to replicate in yeast are the yeast plasmid 2 urn replication genes REP 1 -3 and origin of replication.

[0050] The vectors may contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (ornithine carbamoyltransferase), pyrG

(orotidine-5'-phosphate decarboxylase) and trpC (anthranilate synthase). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. A well suited selectable marker for yeast is the Schizosaccharomyces pombe TPI gene (Russell (1985) Gene 40:125-130).

[0051] In the vector, the polynucleotide sequence is operably connected to a suitable promoter sequence. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intra-cellular polypeptides either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus licheniformis penicillinase gene (penP). Examples of suitable promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid stable alpha-amylase. In a yeast host, useful promoters are the Saccharomyces cerevisiae Mai, TPI, ADH or PGK promoters. The polynucleotide construct will also typically be operably connected to a suitable terminator. In yeast a suitable terminator is the TPI terminator (Alber ei al. (1982) J. Mol. Appl. Genet. 1 :419-434).

[0052] The procedures used to ligate the polynucleotide sequence, the promoter and the terminator, respectively, and to insert them into a suitable vector containing the information necessary for replication in the selected host, are well known to those skilled in the art. It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence encoding a modified CHC polypeptide as described herein, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for individual elements of the CHC followed by ligation.

[0053] Also provided are recombinant host cells, comprising a polynucleotide sequence encoding a modified CHC polypeptide as described herein. A vector comprising such polynucleotide sequence is introduced into the host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Example host cells include bacterial cells, insect cells, mammalian cells, plant cells, and yeast cells.

Pharmaceutical Compositions

[0054] Pharmaceutical compositions comprising a modified CHC polypeptide as described herein can be used in the treatment of conditions which are sensitive to p53-mediated transactivation, such as cancer. Due to their impaired ability to form trimers, the modified CHC polypeptides as described herein can be administered as therapeutic agents without substantially interfering with the formation of vesicles for intercellular transport or other functions of WT clathrin. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modified CHC polypeptide as described herein employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the condition to be treated. It is recommended that the daily dosage of a composition be determined for each individual patient by those skilled in the art in a clinical setting.

[0055] Pharmaceutical compositions of a modified CHC polypeptide as described herein may contain adjuvants and additives typical of pharmaceutical formulations and are usually formulated as an aqueous solution. The aqueous medium may be made isotonic, for example, with sodium chloride, sodium acetate or glycerol. Furthermore, the aqueous medium may contain pH-adjusting additives such as buffers, and preservatives. Consequently, there is also provided a pharmaceutical composition comprising a modified CHC polypeptide as described herein and optionally one or more agents suitable for stabilization, preservation or isotonicity, for example, transition metal ions, phenol, cresol, a parabene, sodium chloride, glycerol or mannitol.

[0056] The pH-adjusting agent used in the pharmaceutical composition may be a buffer selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and

tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.

[0057] The pharmaceutically acceptable preservative may be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2- phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p- hydroxybenzoate, benzethonium chloride, chlorphenesine (3p- chlorphenoxypropane-1 ,2-diol) or mixtures thereof. In some cases, the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In some instances, the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In other instances, the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In further instances, the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0058] The isotonicity agent may be selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. L-glycine, L- histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3-propanediol, 1 ,3- butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.

[0059] Other formulations include suitable delivery forms known in the art including, but not limited to, carriers such as liposomes. See, for example, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomal preparations include, but are not limited to, cytofectins, multilamellar vesicles and unilamellar vesicles. In some aspects, more than one modified CHC as described herein may be administered, for example in compositions that may contain at least one, at least two, at least three, at least four, at least five different modified CHC polypeptides. A mixture of modified CHC polypeptides may be particularly useful in treating a broader range of population of individuals.

[0060] A polynucleotide encoding a modified CHC polypeptide as described herein may also be used for delivery and expression of any of said polypeptides in a desired cell. It is apparent that an expression vector can be used to direct expression of a modified CHC polypeptides according to methods known in the art. The expression vector can be administered by any means known in the art, such as intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, dermally, sublingually, or by inhalation. For example, administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. One skilled in the art is familiar with administration of expression vectors to obtain expression of an exogenous protein in vivo. See, e.g., U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471 .

[0061] Targeted delivery of therapeutic compositions comprising a nucleic acid molecule encoding a modified CHC polypeptide as described herein can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 1 1 :202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol Chem. (1988) 263:621 ; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. ScL (USA) (1990) 87:3655; Wu et al., J Biol. Chem. (1991 ) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol.

[0062] The therapeutic polynucleotides of the present invention can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non- viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1 :51 ; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1 :185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

[0063] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/1 1230; WO 93/10218; WO 91/02805; U.S. Patent Nos. 5,219,740; 4,777,127; GB Patent No. 2,200,651 ; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR- 532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/1 1984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

[0064] Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA(see, e.g., Wu5 J. Biol. Chem. (1989) 264:16985); eukaryotic cell-delivery vehicles cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/1 1092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422, 120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent NO. 0 524 968.

Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:241 1 and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91 :1581 .

[0065] A modified CHC polypeptide is included as active compound in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect. The therapeutically effective concentration can be determined empirically by testing the compounds in known in vitro and in vivo systems. The active compounds can be administered by any appropriate route, for example, orally, nasally, pulmonarily, parenterally, intravenously, intradermally,

subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.

[0066] The modified CHC polypeptide and physiologically acceptable salts and solvates thereof can be formulated for administration by inhalation (either through the mouth or the nose), oral, pulmonary, transdermal, parenteral or rectal administration. For administration by inhalation, the modified CHC can be delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator, can be formulated containing a powder mix of a therapeutic compound and a suitable powder base such as lactose or starch.

[0067] For pulmonary administration to the lungs, the modified CHC can be delivered in the form of an aerosol spray from a nebulizer, turbonebulizer, or microprocessor-controlled metered dose oral inhaler with the use of a suitable propellant. Usually, the particle size of the aerosol spray is small, such as in the range of 0.5 to 5 microns. In the case of a pharmaceutical composition formulated for pulmonary administration, detergent surfactants are not typically used.

[0068] The modified CHC polypeptide can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0069] The modified CHC polypeptide can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).

Formulations for injection can be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder-lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.

[0070] The modified CHC polypeptide can also be formulated for local or topical application, such as for topical application to the skin (transdermal) and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracistemal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, can be formulated as 0.01 %-10% isotonic solutions and pH about 5-7 with appropriate salts. The compounds can be formulated as aerosols for topical application, such as by inhalation.

[0071] The concentration of active compound in a pharmaceutical composition depends on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. The pharmaceutical compositions, if desired, can be presented in a package, in a kit or dispenser device, that can contain one or more unit dosage forms containing the active ingredient. The package, for example, contains metal or plastic foil, such as a blister pack. The pack or dispenser device can be

accompanied by instructions for administration. The pharmaceutical compositions containing the active agents can be packaged as articles of manufacture containing packaging material, an agent provided herein, and a label that indicates the disorder for which the agent is provided.

[0072] For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. , pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. , lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g. , potato starch or sodium starch glycolate); or Wetting agents (e.g. , sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral

administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with Water or other suitable vehicle before use. Such liquid preparations can be prepared by

conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. , sorbitol syrup, cellulose derivatives or hydrogenated edible fats);

emulsifying agents (e.g. , lecithin or acacia); non aqueous vehicles (e.g. , almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. , methyl or propyl p hydroxybenzoates or sorbic acid). The preparations also can contain buffer salts, flavoring, coloring and/or sweetening agents as appropriate.

[0073] Also provided are pharmaceutical compositions of nucleic acid molecules encoding the modified CHC polypeptide and expression vectors encoding them that are suitable for gene therapy. Rather than deliver the protein, nucleic acid molecules can be administered in vivo (e.g., systemically or by other routes), or ex vivo, such as by removal of cells, including lymphocytes, introduction of the nucleic acid molecule therein, and reintroduction into the host or a compatible recipient. Accordingly, modified CHC polypeptide can be delivered to cells and tissues by expression of nucleic acid molecules. The modified CHC polypeptide can be administered as nucleic acid molecules encoding the CHC polypeptide, including ex vivo techniques and direct in vivo expression.

[0074] Nucleic acid molecules can be delivered to cells and tissues by any method known to those of skill in the art. The isolated nucleic acid molecules can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Methods for administering modified CHC polypeptide by expression of encoding nucleic acid molecules include administration of recombinant vectors. The vector can be designed to remain episomal, such as by inclusion of an origin of replication or can be designed to integrate into a chromosome in the cell. Modified CHC polypeptides also can be used in ex vivo gene expression therapy using non-viral vectors. For example, cells can be engineered to express a modified CHC polypeptide, such as by integrating a modified CHC encoding nucleic acid molecule into a genomic location, either operatively linked to regulatory sequences or such that it is placed operatively linked to regulatory sequences in a genomic location. Such cells then can be administered locally or systemically to a subject, such as a patient in need of treatment.

[0075] Viral vectors, include, for example adenoviruses, herpes viruses, retroviruses and others designed for gene therapy can be employed. The vectors can remain episomal or can integrate into chromosomes of the treated subject. A modified CHC polypeptide can be expressed by a virus, which is administered to a subject in need of treatment. Virus vectors suitable for gene therapy include adenovirus, adeno-associated virus, retroviruses, lentiviruses and others noted above. For example, adenovirus expression technology is well-known in the art and adenovirus production and administration methods also are well known. Adenovirus serotypes are available, for example, from the American Type Culture Collection (ATCC, Rockville, MD). Adenovirus can be used ex vivo. For example, cells are isolated from a patient in need of treatment, and transduced with a modified CHC polypeptide-expressing adenovirus vector. After a suitable culturing period, the transduced cells are administered to a subject locally and/or systemically.

Alternatively, modified CHC polypeptide-expressing adenovirus particles are isolated and formulated in a pharmaceutically-acceptable carrier for delivery of a therapeutically effective amount to prevent, treat or ameliorate a disease or condition of a subject. In some situations it is desirable to provide a nucleic acid molecule source with an agent that targets cells, such as an antibody specific for a cell surface membrane protein or a target cell, or a ligand for a receptor on a target cell.

[0076] The nucleic acid molecules can be introduced into artificial chromosomes and other non-viral vectors. Artificial chromosomes, such as ACES (see,

Lindenbaum et al. Nucleic Acids Res. 32(21 ): e172 (2004)) can be engineered to encode and express the isoform. Briefly, mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure using an ACE System. Using this approach, specific loading of one or two gene targets has been achieved in LMTK(— ) and CHO cells.

[0077] In yet another method is a two-step gene replacement technique in yeast, starting with a complete adenovirus genome (Ad2; Ketner et al. Proc. Natl. Acad. Sci. USA 91 : 6186-6190 61 (1994)) cloned in a Yeast Artificial Chromosome (YAC) and a plasmid containing adenovirus sequences to target a specific region in the YAC clone, an expression cassette for the gene of interest and a positive and negative selectable marker.

[0078] The nucleic acids encoding the modified CHC polypeptides can be encapsulated in a vehicle, such as a liposome, or introduced into a cell, such as a bacterial cell, particularly an attenuated bacterium or introduced into a viral vector. For example, when liposomes are employed, proteins that bind to a cell surface membrane protein associated with endocytosis can be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.

[0079] For ex vivo and in vivo methods, nucleic acid molecules encoding the modified CHC polypeptides are introduced into cells that are from a suitable donor or the subject to be treated. Cells into which a nucleic acid molecule can be introduced for purposes of therapy include, for example, any desired, available cell type appropriate for the disease or condition to be treated, including but not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., such as stem cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and other sources thereof.

[0080] For ex vivo treatment, cells from a donor compatible with the subject to be treated or the subject to be treated cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the subject. Treatment includes direct administration, such as, for example, encapsulated within porous membranes, which are implanted into the patient. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes and cationic lipids (e.g., DOTMA, DOPE and DCChol) electroporation, microinjection, cell fusion, DEAE-dextran, and calcium phosphate precipitation methods. Methods of DNA delivery can be used to express modified CHC polypeptides in vivo. Such methods include liposome delivery of nucleic acids and naked DNA delivery, including local and systemic delivery such as using

electroporation, ultrasound and calcium-phosphate delivery. Other techniques include microinjection, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer and spheroplast fusion.

[0081] In vivo expression of a modified CHC polypeptide can be linked to expression of additional molecules. For example, expression of a modified CHC polypeptide can be linked with expression of a cytotoxic product such as in an engineered virus or expressed in a cytotoxic virus. Such viruses can be targeted to a particular cell type that is a target for a therapeutic effect. The expressed modified CHC polypeptide can be used to enhance the cytotoxicity of the virus. In vivo expression of a modified CHC polypeptide can include operatively linking a modified CHC polypeptide encoding nucleic acid molecule to specific regulatory sequences such as a cell-specific or tissue-specific promoter. Modified CHC polypeptides also can be expressed from vectors that specifically infect and/ or replicate in target cell types and/or tissues. Inducible promoters can be use to selectively regulate modified CHC polypeptide expression.

[0082] Nucleic acid molecules in the form of naked nucleic acids or in vectors, artificial chromosomes, liposomes and other vehicles can be administered to the subject by systemic administration, topical, local and other routes of administration. When systemic and in vivo, the nucleic acid molecule or vehicle containing the nucleic acid molecule can be targeted to a cell. Administration also can be direct, such as by administration of a vector or cells that typically targets a cell or tissue. For example, tumor cells and proliferating can be targeted cells for in vivo expression of modified CHC polypeptides. Cells used for in vivo expression of a modified CHC polypeptide also include cells autologous to the patient. These cells can be removed from a patient, nucleic acids for expression of a modified CHC polypeptide introduced, and then administered to a patient such as by injection or engraftment.

[0083] Polynucleotides and expression vectors provided herein can be made by any suitable method. Further provided are nucleic acid vectors containing nucleic acid molecules as described above, including a nucleic acid molecule containing a sequence of nucleotides that encodes the polypeptide as set forth in any of SEQ ID NOS: 3-4 or a functional fragment thereof. Further provided are nucleic acid vectors containing nucleic acid molecules as described above and cells containing these vectors.

Therapeutic Uses

[0084] The modified CHC polypeptides and nucleic acid molecules provided herein can be used for treatment of any condition for which p53-mediated transactivation is employed. This section provides exemplary uses of modified CHC polypeptides and administration methods. Such methods include, but are not limited to, methods of treatment of physiological and medical conditions described and listed below.

[0085] In particular, the modified CHC polypeptides are intended for use in therapeutic methods for the treatment of cancer. Specific examples of cancers include bladder cancer, brain cancer, head and neck cancer, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, myeloma, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, colon cancer, cervical carcinoma, breast cancer, and leukemias. Moreover, because of its ability to promote cancer suppressor p53-mediated transactivation, the modified CHC's are especially well suited for treatment of solid tumors and advanced cancers which cannot be treated by most conventional cancer therapies. The modified CHC polypeptides and nucleic acid molecules encoding modified CHC polypeptides also can be

administered in combination with other therapies including other biologies and small molecule compounds.

[0086] Treatment of diseases and conditions with modified CHC polypeptides can be effected by any suitable route of administration using suitable formulations as described herein, including but not limited to, subcutaneous injection, oral, nasal, pulmonary and transdermal administration. If necessary, a particular dosage and duration and treatment protocol can be empirically determined or extrapolated. For example, exemplary doses of modified CHC polypeptides can be used as a starting point to determine appropriate dosages. Particular dosages and regimens can be empirically determined.

[0087] Dosage levels would be apparent to one of skill in the art and would be determined based on a variety of factors, such as body weight of the individual, general health, age, the activity of the specific compound employed, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease or condition, and the subject's disposition to the disease/condition and the judgment of the treating physician. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form with vary depending upon the subject treated and the particular mode of administration.

Upon improvement of a subject' s condition, a maintenance dose of a compound or composition provided herein can be administered, if necessary; and the dosage, the dosage form, or frequency of administration, or a combination thereof, can be varied. In some cases, the subject can require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

[0088] Administration of a modified CHC polypeptide in accordance with the methods of the present invention can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of may be essentially continuous over a

preselected period of time or may be in a series of spaced dosages, e.g., either before, during, or after the insurgence of cancers.

[0089] Also provided herein is the use of any of the modified CHC polypeptides provided herein for the manufacture of a medicament for treating of a subject having cancer. Specific examples of cancers include by way of example bladder cancer, brain cancer, head and neck cancer, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, myeloma, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, colon cancer, cervical carcinoma, breast cancer, and leukemias.

EXPERIMENTAL

[0090] A clathrin fragment (designated CTXD, aa1521 -1654 of SEQ ID NO:2) capable of trimerization (Figure 1 ) that complemented previously solved domains of clathrin was synthesized [10, 33]. The CTXD construct did not have a light chain- binding site and featured two point mutations (C1528A and T1585L) to increase protein stability and solubility. At first, the quality of CTXD crystals was

disappointing because they would not diffract beyond approximately 9 A. However, after evaluating hundreds of crystallization conditions, small molecule additives, and detergents, it was found that a brief soak in sodium bromide dramatically increased diffraction to approximately 3.9 A.

[0091] Synchrotron data were collected on the best crystals (Advanced Light Source MBC 4.2.2) and the structure was solved by molecular replacement. The atomic coordinates have been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank PDB (http://www.pdb.org, PDB code 3QIL). The structure in Figure 1 has the recognizable features of the trimerization domain in lower resolution models [13, 15], but there are noticeable differences. The green leg in Figure 1 is slipped down and away from the grey legs, indicating the 3-fold symmetry of the trimerization domain is distorted, which is consistent with the fact that CTXD does not have any light chain to add stability [25]. This finding is supported by small-angle neutron scattering experiments showing the trimerization region is highly flexible [34]. The CTXD structure also suggests the long tripod helices, which have been modeled previously as straight and rigid [13, 15], have bends in the CTXD structure. It is not believed these bends are the result of model building bias because Rf_ree worsens when they are made straight. After glutamate-1624, the long helix abruptly changes direction, but the remaining 30 residues (1625-1654) could not be built because of weak electron density. This C- terminal region contains the i638QLMLTi₆₄₂ sequence that closely resembles the consensus sequence [35] that binds the uncoating ATPase Hsc70 [36]. The idea that natively unfolded proteins [37, 38] are important in endocytosis [39] raises the possibility that the C-terminal ends of heavy chains are disordered to capture auxilin and begin Hsc70-dependent lattice disassembly [40, 41 ].

[0092] Finally, it was observed that helix 7g (aa1521 -1529) in each of the three legs of CTXD is completely unfolded (see asterisk in Figure 1 ). This implies any favorable contact with neighboring and natively folded helix 7h (aa1534-1543), is insufficient to prevent helix 7g from unraveling. Therefore, this region of the proximal domain may be inherently flexible to facilitate the self-assembly of clathrin. This observation is consistent with findings that the clathrin heavy chain is modestly flexible [34, 42, 43].

Identifying a Topology Switch

[0093] Differences are revealed when a leg from assembled clathrin or clathrin hub (both with bound light chains) is superimposed over a leg from CTXD. First, the short Tx2 helix (dark grey in Figure 2A) in assembled clathrin [15] and clathrin hub [13] is unfolded in every leg of CTXD. This is potentially significant because Ambivalent Structure Predictor (ASP) [44] predicts isonN IMDFAMPisg? (Tx2 underlined) is a conformational switch (labeled pCS in Figure 2). Second, helix 7j is shifted away from 1590N IMDFAMP-1597 (see arrow, Figure 2A) towards helix 7h (residue 1532-1544). Helix 7j is displaced by approximately 10 A relative to the grey helix in Figure 2A taken from assembled clathrin (PDB code 1 X14).

Interestingly, helix 7j from the clathrin hub (PDB code 3LVG) [13] lies in between the indicated helices in Figure 2A. To confirm position, helix 7j was omitted from each of the 24 protomers in the asymmetric unit. Calculated F₀ - F_c maps returned electron density where helix 7j was originally located. The stability of Tx2 in 1590N IM DFAMP-1597 may be supported by helix 7j because Tx2 is unraveled when helix 7j is out of reach (Figure 2A).

[0094] It is pointed out that glycine-1567 is positioned near the middle of helix 7j, which is noteworthy because glycine is known to be a helix breaker [45]. The assembled clathrin and clathrin hub models are both saturated with light chains, but CTXD does not have any. Without being bound to any particular theory, the fact that helix 7j is offset a distance approximately equal to the diameter of a helix raises the possibility that the position of helix 7j is subject to the light chain. This suggestion is supported by the observation that C-terminal segments of the light chain are close to helix 7j [13]. The structural components in Figure 2 are conserved in lower and higher invertebrates and vertebrates, suggesting they may be relevant for function.

[0095] The daisy-wheel organization of contact surfaces in the topology switch model presented in Figure 2 offers a conceptual framework for previous findings that cysteine-1573 is vital for clathrin stability [25, 46]. In the switch model, the physical contact between helix 7j and 1590N IMDFAMP1597 (see red/yellow contacts, Figure 2C) forms the basis for the interaction surface that brings neighboring legs together. The CTXD model suggests moving helix 7j away distorts the trimer by affecting red/yellow contacts (see Figs. 1 and 2). However, a second interaction surface defined by the helix tripod must also keep legs together because CTXD is trimeric in the crystal.

[0096] This observation may be partially explained by the switch model because cysteine-1573 is located in helix 7j (Figure 2). However, any influence of cysteine- 1573 on the helix tripod is likely to be indirect because the core of the helix tripod is far in space from cysteine-1573. The working switch model indicates two red/yellow contacts must break to free a single leg. A by-product of this step is a clathrin dimer. To finish converting this dimer to monomers, only one red/yellow contact needs to break, suggesting there is built-in cooperativity if the process is in fact step-wise. The model predicts the action of one switch in a leg is sufficient to modify the geometry of the trimerization domain. This may be informative for interpreting Monte Carlo simulations that show the formation of clathrin baskets is sensitive to adjustments in the pucker of legs radiating from the trimerization domain [47, 48].

Nuclear Localization is Mediated by a Conserved Cysteine in Helix 7j.

[0097] Clathrin participates in activating cancer suppressor p53 in the nucleus when clathrin is made monomeric [31 ]. To determine if clathrin detrimerization occurs in the cytosol or in the nucleus, non-cancer (HEK293T) and cancer (HeLa, H1299, and MCF7) cells were transfected with human influenza hemagglutinin (HA)-tagged WT trimeric hub or with HA-C1573A hub and observed by means of confocal microscopy. The ability of clathrin hub to enter the nucleus clearly depends on cysteine-1573 (Figure 3). In all cell types tested, the nuclear localization of clathrin hub significantly increases when cysteine-1573 is changed to alanine, compared to WT (Figure 3A).

[0098] The microscopy results are confirmed by Western blots of

cytosolic/membrane and nuclear fractions (Figure 3B), which show HA-positive signal in nuclear fraction of cells expressing HA-C1573A hub only. To see if the conserved histidine before 1590N IMDFAMP-1597 plays a role in subcellular clathrin hub localization, cells expressing HA-H1589C hub were visualized. It was observed that this mutant has an intermediate effect that is slightly amplified in HeLa, H1299, and MCF7 cancer cells (see Figure 3A). This suggests histidine-1589 has a role to play, but not as important as cysteine-1573.

[0099] The structure of the trimerization domain illustrated herein shows cysteine-1573 is located in helix 7j, which is part of a topology switch that includes the 1590N IMDFAM P-1597 segment that is predicted to undergo a conformational change. The evolutionarily conserved components in Figure 2 are uniquely organized to form a chain of interfaces that connect legs together in the trimerization domain. Microscopy data show HA-tagged WT hub is exclusively

cytosolic/membrane in non-cancer and cancer cells while HA-tagged C1573A hub accumulates inside the nucleus. Together, the structure and microscopy

experiments here establish a sensitive helix is involved in the destabilization of trimeric clathrin to yield individual legs in the cytosol for localization into the nucleus. This localization is believed to be essential for clathrin to interact with the p53 pathway to suppress tumorigenesis.

Dynamic Light Scattering

[00100] To determine if the overall size of trimeric clathrin was sensitive to cysteine-1573, dynamic light scattering (DLS) measurements were performed on WT clathrin hub, C1573A hub, and a clathrin control that did not have a trimerization domain (aa865-1521 ). Table 1 presents three independent DLS experiments for each clathrin construct. For any given DLS run, data were recorded 13 times to ensure consistency. The average particle size according to DLS of C1573A hub (18.30±1 .35 nm) was smaller than WT hub (22.94±0.93 nm). For comparison, the mean size of the monomer control, clathrin 865-1521 , was 15.69±2.67 nm.

Together, the DLS data indicate the mean particle size of clathrin hub is reduced by -20% when cysteine-1573 in helix 7j is replaced with alanine. The fact that the average particle size of C1573A hub estimated by DLS is similar to that of 865-1521 clathrin suggests the measured difference is significant. These data are consistent with the in-cell observations that mutating cysteine-1573 in the sensitive helix of the topology switch converts trimeric clathrin into a monomer.

Table 1 : Clathrin particle size bv dynamic liqht scatterinq

aAverages and standard deviations were determined from 3 independent experiments for each construct. 1 nm equals 10^"9 meters.

[00101] In view of the above, it will be seen that several advantages of the compositions and methods as described herein are achieved and other

advantageous results attained. Not all of the depicted components illustrated or described may be required. In addition, some implementations and embodiments may include additional components. Variations in the arrangement and type of the components may be made without departing from the spirit or scope of the claims as set forth herein. Additional, different or fewer components may be provided and components may be combined. Alternatively or in addition, a component may be implemented by several components.

[00102] The above description illustrates compositions and methods by way of example and not by way of limitation. This description clearly enables one skilled in the art to make and use the compositions and methods as described herein, and describes several embodiments, adaptations, variations, alternatives and uses thereof. Additionally, it is to be understood that the compositions and methods are not limited in their application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. Other embodiments may be practiced or carried out in various ways. Also, it will be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[00103] Having described aspects of the disclosure in detail, it will be apparent that modifications and variations are possible without departing from the scope of such aspects as defined in the appended claims. As various changes could be made in the above compositions and methods without departing from the scope of the aspects described herein, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. MATERIALS AND METHODS

[00104] Crystals. pET15b encoding bovine fragment 1521 -1654 (CTXD, C1528Α and T1585L mutations for solubility) with an N-terminal histidine-tag was

constructed using standard cloning methods. Rosetta 2(DE3) plysS cells with CTXD were grown at 37 °C in LB and induced at 30 °C with IPTG. Cell pellets were resuspended and sonicated in cold lysis buffer (10 mM Na₂HP0₄, 10 mM imidazole, 0.5 M NaCI, pH 7.4 with Triton X-100, β-mercaptoethanol, and protease inhibitors). Crude lysate was passed through a metal affinity column (GE Healthcare), and a POROS 20 HQ anion-exchange column (Perseptive Biosystems) at 4 °C

equilibrated with 10 mM HEPES, 20 mM imidazole, 1 % (v/v) glycerol, 10 mM β- mercaptoethanol at pH 8.5. Finally, the protein was polished using a Superdex 200 column. Protein at 25 mg/ml was crystallized by the hanging drop method in 2.0 M NaCI, 80mM imidazole, pH 7.0 with some PEG 3350. Crystals grew in the trigonal space group R3 (a = 255.78, b = 255.78, c = 312.99, and a = 90°, β = 90°, γ = 120°, hexagonal setting), with 24 protomers (8 trimers) in the asymmetric unit. Diffraction was significantly improved by soaking 2-3 week old crystals in 87.7 mM imidazole, 36% (v/v) glycerol cryogenic buffer containing n-tetradecyl-p-D-maltoside for 20 minutes exposed to open air before plunging in liquid nitrogen.

[00105] Data collection. A data set was collected at 100 °K in 0.3° oscillations using a NOIR-1 CCD detector at the MBC 4.2.2 beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory. Data were processed using HKL2000.

[00106] Phasing and refinement. Crystal data were phased by molecular replacement using Phaser [49]. A trimer model from the cryo-EM structure (PDB code 1 X14) failed, but a solution was found using search models with manually altered leg orientations. The model was built with O [50] and refined using CNS [51 , 52]. Refinement was carried out on partially twinned data (twinning operators = k, h, -I and twinning fraction = 0.484, determined by CNS). Because there were 24 protomers in the asymmetric unit, non-crystallographic symmetry (NCS) restraints were used throughout the refinement. To minimize distortions, simulated annealing was performed with harmonically restrained backbone atoms (harmonic restraint constant = 10). This was interspersed with model rebuilding with reference to 2F₀ - F_c and F₀ - F_c maps. Electron density of the histidine tags (not in the search model) of two legs became visible in 2F₀ - F_c maps. The clathrin trimerization domain structure refined against all the data with an R_work of 33.9% and an R_free of 38.7% at 3.9 A resolution.

[00107] Figure 1 , left, domain map of three identical heavy chain legs (A, B, and C) of clathrin: NTD, N-terminal domain; DD, distal domain; PD, proximal domain (light chain binding); CTXD, trimerization domain. CTXD construct (aa1521 -1654, red bar) with an N-terminal histidine-tag associates into a trimer. Right, green leg C (numbered 1 -1624) is tilted down and away from the two grey legs. Histidine tags are visible in legs A and C, but not leg B. Residues 1625-1654 were not modeled, but packing of 24 protomers in the asymmetric unit leaves room for them. Asterisk indicates unfolded helix 7g in the green colored leg (this helix is also disordered in the other two legs).

Identification of a topology switch (Figure 2).

[00108] Figure 2A, Helix 7j (magenta) and predicted conformational switch (pCS, colored red) in Leg B (PDB 3QIL, salmon color) is compared to the same structures in PDB 1 X14 [15] (green) and PDB 3LVG [13] (blue, only Helix 7j shown for clarity). A one-turn helix (Tx2 in 1 X14, colored green) is unfolded in 3QIL (red strand).

Labeled residues indicate the register of the superposition. B, Top view shows the spatial arrangement of Helix 7j, Tx1 , and pCS (color coding the same as in A).

Cysteine-1573 (pale teal) is adjacent to pCS marked by histidine-1589 (salmon, asterisk in D). C, Switch model shows how molecular contacts between pCS (red) and Tx1 (yellow) in the next leg over are mediated by helix 7j/pCS interactions (curved arrows). The numbers refer to the sequence that releases a leg. Two switches must be activated to release one monomer. D, The putative topology switch is conserved in lower and higher vertebrates and invertebrates. Cysteine- 1573 (teal star) is almost entirely conserved compared to other cysteines (black diamonds) in this helix (note: the human clathrin sequence NP_004850.1 in the sequence alignment has a different numbering scheme than the bovine clathrin sequence).

Nuclear localization of clathrin hub following mutations in helix 7j (Figure 3).

[00109] All reagents from Gibco (Invitrogen) unless indicated otherwise. Cells were maintained at 37 °C, in a 5% C02 humidified incubator. HeLa (cervical cancer) and HEK293T (human embryonic kidney) cells were maintained in DMEM with 10% fetal bovine serum (FBS, Sigma-Aldrich) 100 U penicillin/100 μg streptomycin. H1299 (non-small cell lung cancer) cells were maintained in RPMI- 1640 media with 10% FBS, 10 mM HEPES, 100 U penicillin/100 streptomycin, 2 mM Glutamax, and 1 mM sodium pyruvate. MCF7 (breast cancer) cells were maintained in MEM with 10% FBS, 100 U penicillin/100 μg streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 % sodium bicarbonate, and 120 ng/ml insulin (Sigma).

[00110] Confocal microscopy. H1299, MCF7, HeLa, and 293T cells were plated onto poly-l-lysine coated coverslips and transfected with pcDNA3.1 (-)HA-hub (1074- 1675) WT, C1573A, or H1589C plasmid DNA using FugeneHD (Roche). After 24 hours, cells were fixed, permeabilized, and stained with anti-HA (Covance) and anti- mouse AlexaFluor 488 (Molecular Probes, Invitrogen) antibodies. Cells were counterstained with Draq5 (Cell Signaling Technologies) and coverslips mounted using ProLong Gold (Invitrogen). Images were obtained using a Leica Sp5 scanning confocal system. A minimum of 100 cells over four independent experiments were quantified using Leica LAF software. Any signal within the nucleus above background was scored nuclear positive. Statistical significance was determined using one-way ANOVA.

[00111] Subcellular fractionation. Cells were plated in six well plates and transfected as for confocal experiments. After 24 hours, cytosolic/membrane and nuclear fractions were prepared. Fractions were loaded equally onto 10% SDS PAGE gels and Western blotted using anti-HA (Covance) and nuclear-specific anti- TATA binding protein (Abeam) antibodies to confirm fractionation. [00112] Figure 3. A, The percentage of cells with nuclear signal is much higher (over 90%) in cells expressing HA-hub C1573A compared to HA-hub WT or HA-hub H1589C cells. Error bars are SEM. ***p<0.001 . B, HA signal is detectable in the nuclear-positive fractions of H1299 cells. Western blotted with anti-HA and anti- TATA binding protein (TBP)-a specific nuclear marker. HA signal is present in the cytoplasmic fractions of both HA-hub WT and HA-hub C1573A, as well as in the nuclear fraction of cells expressing HA-hub C1573A, but not HA-hub WT.

[00113] Dynamic light scattering measurements. Purified WT hub, C1573A hub an clathrin 865-1521 were concentrated to 0.3 mg/ml. Samples were spun at 15,000 rpm, 4 degrees Celsius for 5-10 minutes just before use. Measurements were performed at room temperature on a Malvern Zetasizer Nano-S. Experiments were done on three independent protein preparations for each construct.

REFERENCES

1 . Kirchhausen, T. (1999). Adaptors for clathrin-mediated traffic. Annu. Rev. of Cell and Dev. Biol. 15, 705-32.

2. Brodsky, F. M., Chen, C. Y., Knuehl, C, Towler, M. C. & Wakeham, D. E. (2001 ). Biological basket weaving: formation and function of clathrin-coated vesicles. Annu Rev Cell Dev Biol 17, 517-68.

3. Robinson, M. S. (2004). Adaptable adaptors for coated vesicles. Trends Cell Biol 14, 167-74.

4. Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H. & Benovic, J. L. (1996). b-arrestin acts as a clathrin adaptor in endocytosis of the b2-adrenergic receptor. Nature 383, 447-450.

5. Krupnick, J. G., Goodman Jr., O. B., Keen, J. H. & Benovic, J. L. (1997). Arrestin-clathrin interaction. Localization of the clathrin binding domain of nonvisual arrestins to the carboxyl terminus. J. Biol. Chem. 272, 1501 1 -15016.

6. Rothman, J. E. & Schmid, S. L. (1986). Enzymatic recycling of clathrin from coated vesicles. Cell 49, 5-9.

7. Hannan, L. A., Newmyer, S. L. & Schmid, S. L. (1998). ATP- and cytosol- dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for Hsc70. Molecular Biology of the Cell 9, 2217-29. 8. Ma, Y., Greener, T., Pacold, M. E., Kaushal, S., Greene, L. E. & Eisenberg, E. (2002). Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70. J Biol Chem 277, 49267-74.

9. Heymann, J. B., Iwasaki, K., Yim, Y. I., Cheng, N., Belnap, D. M., Greene, L. E., Eisenberg, E. & Steven, A. C. (2005). Visualization of the binding of Hsc70

ATPase to clathrin baskets: implications for an uncoating mechanism. J Biol Chem 280, 7156-61 .

10. ter Haar, E., Musacchio, A., Harrison, S. C. & Kirchhausen, T. (1998). Atomic structure of clathrin: A b propeller terminal domain joins an a zigzag linker. Cell 95, 563-573.

1 1 . ter Haar, E., Harrison, S. C. & Kirchhausen, T. (2000). Peptide-in-groove interactions link target proteins to the beta-propeller of clathrin. Proc Natl Acad Sci U S A 97, 1096-100.

12. Ybe, J. A., Brodsky, F. M., Hofmann, K., Lin, K., Liu, S. H., Chen, L., Earnest, T. N., Fletterick, R. J. & Hwang, P. K. (1999). Clathrin self-assembly is mediated by a tandemly repeated superhelix. Nature 399, 371 -5.

13. Wilbur, J. D., Hwang, P. K., Ybe, J. A., Lane, M., Sellers, B. D., Jacobson, M. P., Fletterick, R. J. & Brodsky, F. M. Conformation switching of clathrin light chain regulates clathrin lattice assembly. Dev Cell 18, 841 -8.

14. Liu, S.-H., Wong, M. L, Craik, C. S. & Brodsky, F. M. (1995). Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs. Cell 83, 257-267.

15. Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S. C, Kirchhausen, T. & Walz, T. (2004). Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-9.

16. Kirchhausen, T., Harrison, S. C, Parham, P. & Brodsky, F. M. (1983).

Location and distribution of the light chains in clathrin trimers. Proc. Natl. Acad. Sci. USA 80, 2481 -2485.

17. Kirchhausen, T., Scarmato, P., Harrison, S. C, Monroe, J. J., Chow, E. P., Mattaliano, R. J., Ramachandran, K. L., Smart, J. E., Ahn, A. H. & Brosius, J.

(1987b). Clathrin light chains LCa and LCb are similar, polymorphic, and share repeated heptad motifs. Science 236, 320-324. 18. Brodsky, F. M., Hill, B. L, Acton, S. L, Nathke, I., Wong, D. H.,

Ponnambalam, S. & Parham, P. (1991 ). Clathrin light chains: Arrays of protein motifs that regulate coated vesicle dynamics. Trends Biol. Sci. 16, 208-213.

19. Newpher, T. M., Idrissi, F. Z., Geli, M. I. & Lemmon, S. K. (2006). Novel function of clathrin light chain in promoting endocytic vesicle formation. Mol Biol Cell 17, 4343-52.

20. Chen, C. Y. & Brodsky, F. M. (2005). Huntingtin-interacting protein 1 (Hip1 ) and Hip1 -related protein (HipI R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo. J Biol Chem 280, 6109-17.

21 . Legendre-Guillemin, V., Metzler, M., Lemaire, J. F., Philie, J., Gan, L, Hayden, M. R. & McPherson, P. S. (2005). Huntingtin interacting protein 1 (HIP1 ) regulates clathrin assembly through direct binding to the regulatory region of the clathrin light chain. J Biol Chem 280, 6101 -8.

22. Niu, Q. & Ybe, J. A. (2008). Crystal structure at 2.8 A of Huntingtin-interacting protein 1 (HIP1 ) coiled-coil domain reveals a charged surface suitable for HIP1 protein interactor (HIPPI). J Mol Biol 375, 1 197-205.

23. Huang, K. M., Gullberg, L, Nelson, K. K., Stefan, C. J., Blumer, K. &

Lemmon, S. K. (1997). Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast. J Cell Sci 1 10 (Pt 7), 899-910.

24. Wang, J., Wang, Y. & O'Halloran, T. J. (2006). Clathrin light chain:

importance of the conserved carboxy terminal domain to function in living cells. Traffic 7, 824-32.

25. Ybe, J. A., Perez-Miller, S., Niu, Q., Coates, D. A., Drazer, M. W. & Clegg, M. E. (2007). Light chain C-terminal region reinforces the stability of clathrin heavy chain trimers. Traffic 8, 1 101 -10.

26. Royle, S. J., Bright, N. A. & Lagnado, L. (2005). Clathrin is required for the function of the mitotic spindle. Nature 434, 1 152-7.

27. Royle, S. J. & Lagnado, L. (2006). Trimerisation is important for the function of clathrin at the mitotic spindle. J Cell Sci 1 19, 4071 -8.

28. Young, J. S., Guttman, J. A., Vaid, K. S., Shahinian, H. & Vogl, A. W. (2009). Cortactin (CTTN), N-WASP (WASL), and clathrin (CLTC) are present at podosome- like tubulobulbar complexes in the rat testis. Biol Reprod 80, 153-61 . 29. Blixt, M. K. & Royle, S. J. Clathrin heavy chain gene fusions expressed in human cancers: analysis of cellular functions. Traffic 12, 754-61 .

30. Enari, M., Ohmori, K., Kitabayashi, I. & Taya, Y. (2006). Requirement of clathrin heavy chain for p53-mediated transcription. Genes Dev 20, 1087-99.

31 . Ohmori, K., Endo, Y., Yoshida, Y., Ohata, H., Taya, Y. & Enari, M. (2008). Monomeric but not trimeric clathrin heavy chain regulates p53-mediated

transcription. Oncogene 27, 2215-27.

32. Ohata, H., Ota, N., Shirouzu, M., Yokoyama, S., Yokota, J., Taya, Y. & Enari, M. (2009). Identification of a function-specific mutation of clathrin heavy chain (CHC) required for p53 transactivation. J Mol Biol 394, 460-71 .

33. Ybe, J. A., Brodsky, F. M., Hofmann, K., Lin, K., Liu, S.-H., Chen, L., Earnest, T. N., Fletterick, R. J. & Hwang, P. K. (1999). Clathrin self-assembly is mediated by a tandemly repeated superhelix. Nature 399, 371 -375.

34. Ferguson, M. L., Prasad, K., Boukari, H., Sackett, D. L., Krueger, S., Lafer, E. M. & Nossal, R. (2008). Clathrin triskelia show evidence of molecular flexibility.

Biophys J 95, 1945-55.

35. Gragerov, A., Zeng, L., Zhao, X., Burkholder, W. & Gottesman, M. E. (1994). Specificity of DnaK-peptide binding. J Mol Biol 235, 848-54.

36. Hartl, F. U. & Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852-8.

37. Dunker, A. K., Brown, C. J., Lawson, J. D., lakoucheva, L. M. & Obradovic, Z. (2002). Intrinsic disorder and protein function. Biochemistry 41 , 6573-82.

38. Ward, J. J., Sodhi, J. S., McGuffin, L. J., Buxton, B. F. & Jones, D. T. (2004). Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J Mol Biol 337, 635-45.

39. Dafforn, T. R. & Smith, C. J. (2004). Natively unfolded domains in

endocytosis: hooks, lines and linkers. EMBO Rep 5, 1046-52.

40. Rapoport, I., Boll, W., Yu, A., Booking, T. & Kirchhausen, T. (2008). A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction. Mol Biol Cell 19, 405-13.

41 . Xing, Y., Booking, T., Wolf, M., Grigoheff, N., Kirchhausen, T. & Harrison, S. C. Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70- facilitated disassembly. Embo J 29, 655-65. 42. Jin, A. J. & Nossal, R. (2000). Rigidity of triskelion arms and clathrin nets. Biophys J 78, 1 183-94.

43. Kotova, S., Prasad, K., Smith, P. D., Lafer, E. M., Nossal, R. & Jin, A. J. AFM visualization of clathrin triskelia under fluid and in air. FEBS Lett 584, 44-8.

44. Young, M., Kirshenbaum, K., Dill, K. A. & Highsmith, S. (1999). Predicting conformational switches in proteins. Protein Sci 8, 1752-64.

45. Chakrabartty, A., Kortemme, T. & Baldwin, R. L. (1994). Helix propensities of the amino acids measured in alanine-based peptides without helix-stabilizing side- chain interactions. Protein Sci 3, 843-52.

46. Ybe, J. A., Ruppel, N., Mishra, S. & VanHaaften, E. (2003). Contribution of cysteines to clathrin trimerization domain stability and mapping of light chain binding. Traffic 4, 850-6.

47. den Otter, W. K., Renes, M. R. & Briels, W. J. Self-assembly of three-legged patchy particles into polyhedral cages. J Phys Condens Matter 22, 104103.

48. den Otter, W. K., Renes, M. R. & Briels, W. J. Asymmetry as the key to clathrin cage assembly. Biophys J 99, 1231 -8.

49. McCoy, A. J. (2007). Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D Biol Crystallogr 63, 32-41 .

50. Jones, T. A., Bergdoll, M. & Kjeldgaard, M. (1990). O: a macromolecule modelling environment. In Crystallography and Modelling Methods in Molecular

Design (Bugg, C. & Ealick, S., eds.), pp. 189-199. Springer- Verlag, New York.

51 . Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L, Gros, P., Grosse- Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. & Warren, G. L. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta

Crystallographica. Section D: Biological Crystallography 54, 905-21 .

52. Brunger, A. T. (2007). Version 1 .2 of the Crystallography and NMR system. Nat Protoc 2, 2728-33.