Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
1998-06-19
2001-09-25
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S183000, C435S219000, C435S226000
Reexamination Certificate
active
06294363
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of proteolytic degradation of cellular proteins. More specifically, rapid and efficient methods for proteasome purification from various cell types are disclosed. Also provided are novel methods for use of the proteasome components so purified.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application by author name and year of publication in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
The degradation of cellular proteins is necessary for the biological well-being of all organisms. Regulators of cell growth and development, and components of the immune and cellular defense mechanisms are regulated by proteolysis. Membrane receptors and transcription factors activated by cytokines, such as interleukins and interferons, are regulated by protein degradation.
The major pathway of intracellular proteolysis involves the ubiquitin/proteasome system. Ubiquitin, a 76 amino acid polypeptide, is the most highly conserved protein in eukaryotic evolution. There are only 3 amino acid differences between yeast and human ubiquitins. Extensive studies during the past decade have shown that the covalent attachment of ubiquitin to cellular proteins marks them for destruction. Substrates that are linked to ubiquitin are degraded by a multicatalytic protease called the proteasome. During the past few years many targets of the ubiquitin/proteasome system have been discovered and remarkably they include a broad range of regulators of cell growth. Some of the proteins destroyed by the ubiquitin/proteasome system include cyclins, cyclin-dependent kinases (CDK's), NFkB, IkB&agr;, cystic fibrosis transduction receptor, p53, ornithine decarboxylase (ODC), 7-membrane spanning receptors, Cdc25 (phosphotyrosine phosphatase), Rb, G&agr;, c-Jun and c-Fos.
The ubiquitin/proteasome pathway is also essential for the stress-response and for the generation of antigenic peptides in MHC Class I molecules. It is clear that defects in the functioning of the ubiquitin/proteasome system can have severe consequences on biological homeostasis. Indeed, mutations that affect the degradation of many of the proteins listed above have been associated with tumorigenesis.
The 26S-proteasome comprises two distinct sub-complexes. The core complex has a sedimentation velocity of 20S and contains a variety of degradative activities. The 20S core is highly conserved across evolutionary distance and consists of a barrel of 4 rings. Each ring contains 7 subunits of either &agr; class or &agr; class. The rings are oriented so that two &agr;-subunit-containing rings are on the outside, while two &bgr;-subunit containing rings are juxtaposed on the inside. Thus, the 20S core is identical at its two ends. The x-ray structure of the archaebacterial proteasome has recently been resolved and was shown to contain a narrow pore in each &agr; ring, and a large central cavity formed by the &bgr; rings. Accordingly, the central cavity is not exposed to the cellular environment, thereby preventing non-specific degradation of cellular proteins. Proteins targeted for degradation are first threaded through the narrow pores in the a rings before they gain access to the central catalytic cavity.
The second sub-complex, referred to as the 19S-regulatory complex, binds to the ends of the 20S core and regulates access of cellular proteins to the catalytic cavity. The 19S complex, together with the 20S core make up the 26S-proteasome. The 19S complex has at least 6 distinct ATPase subunits which are thought to promote unfolding of proteolytic substrates so that they can be channeled through the narrow pores of the 20S core. The 19S complex contains as many as 20 subunits, which include a multiubiquitin-chain binding protein, isopeptidases and at least 6 ATPases. To date, many of these additional subunits remain uncharacterized.
The Rad23 gene of
S. cerevisiae
is necessary for efficient nucleotide excision repair of damaged DNA. In vitro studies indicate that this factor may play a role in assisting the assembly of the repair complex at the site of damage. Accordingly, interactions between Rad23p and other repair proteins including Rad4p, Rad14p, and subunits of TFIIH have been proposed. Thus far, however, the exact biochemical function of Rad23p in DNA repair has remained unclear.
Rad23p has an NH
2
-terminal domain with striking homology to ubiquitin (22% identity, 43% homology). Watkins et al. have shown that this ubiquitin-like domain is required for repair activity of the protein and that the domain can be replaced by the sequence of wild-type ubiquitin. In addition, a family of proteins with similar ubiquitin-like domains have been discovered. Unfortunately, these family members have diverse species of origin and apparently disparate functions and thus have provided no clue as the exact role of this domain.
As noted above, impaired activity of the proteasome is implicated in many diseases in humans. This observation has stimulated considerable research activity in the identification of novel therapeutic agents for inhibiting and/or stimulating the activity of the proteasome. These studies have been hindered by the inefficient, time-consuming, biochemical protocols available for the purification of proteasomes. The present invention describes a rapid and efficient proteasome purification method and provides novel methods of use of various proteasome subunits so purified.
SUMMARY OF THE INVENTION
The present invention provides compositions and a rapid and efficient method for the purification of proteasome complexes from a variety of cell types. In accordance with the present invention, it has been discovered that the ubiquitin-like N-terminal domain of a yeast protein, Rad23, has high affinity for the proteasome. Accordingly, this domain or homologues thereof may be immobilized to a suitable solid support and used to isolate the proteasome from cell lysates. Following removal of non-specifically bound proteins, the proteasomes are eluted. This method will facilitate the molecular characterization of the as yet unidentified subunits of the proteasome. Ubiquitin-like domains (UbL) in cellular proteins vary slightly between species. In one embodiment of the invention, UbL-domains from a given species will be used for proteasome purification from cell lysates derived from cells of that species.
Another aspect of the invention is a kit of materials useful in performing the proteasome purification method of the invention. A kit according to this aspect of the invention comprises a solid support to which a UbL of interest has been affixed as well as suitable buffers for eluting proteasome preparations.
In a further embodiment of the invention, it has been discovered that this same N-terminal ubiquitin like domain of Rad23, UbL
R23
, functions as a degradation signal in actively growing cells. Fusion proteins comprising this domain are provided herein. Reporter proteins attached to the UbL domain (UbL
R23
-reporter) are rapidly degraded in logarithmically growing cells. Since a primary feature of malignant cells is the aberrant rate of cell growth, the UbL
R23
-reporter provides a powerful way to assess the proliferative potential of tumor cells. In yet another embodiment of the invention, the efficacy of anti-cancer drugs can be assessed by determining the stability of the UbL
R23
-reporter fusion proteins.
In a further aspect of the invention, compositions and methods are provided for enhancing the thermostability of fusion proteins containing the UbL domain. Such fusion proteins may be used to advantage in chemical reactions requiring thermostable reagents, such as the polymerase chain reaction (PCR). In this embodiment of the invention, DNA constructs are generated wherein a DNA sequence encoding a UbL-domain is operably linked to a DNA sequence encoding
Ewing Saul
Nashed Nashaat T.
Reed, Esq. Janet
University of Medicine & Dentistry of New Jersey
Walicka M
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