Methods for diagnosing and treating autoimmune disease

Details Methods for diagnosing and treating autoimmune disease Methods for diagnosing and treating autoimmune disease

1998-02-27

2003-09-09

Nolan, Patrick J. (Department: 1644)

Chemistry: analytical and immunological testing

For preexisting immune complex or auto-immune disease

C435S007100

Reexamination Certificate

active

06617171

ABSTRACT:

FIELD OF THE INVENTION
The invention relates in general to the diagnosis and treatment of immune disorders.
BACKGROUND OF THE INVENTION
Proteolysis in the Cell
A. The Proteasome
In the cytosol, there is a soluble proteolytic pathway that requires ATP and involves covalent conjugation of the cellular proteins with the small polypeptide ubiquitin, or Ub, (Hershko et al., 1992,
Ann. Rev. Biochem.,
61: 761-807; Rechsteiner et al., 1987,
Ann. Rev. Cell. Biol.,
3: 1-30). Thereafter, the conjugated proteins are hydrolyzed by a 26S proteolytic complex containing a 20S degradative particle called the proteasome (Goldberg, 1992,
Eur. J. Biochem.,
203: 9-23); Goldberg et al., 1992,
Nature,
357: 375-379). This multicomponent system is known to catalyze the selective degradation of highly abnormal proteins and short-lived regulatory proteins. However, the system also appears to be responsible for the breakdown of most proteins in maturing reticulocytes (Boches et al., 1982,
Science,
215: 978-980); Spenser et al., 1985,
J. Biol. Chem.,
257: 14122-14127), in growing fibroblasts (Ciechanover et al., 1984,
Cell,
37: 57-66; Gronostajski et al., 1985,
J. Biol. Chem.,
260: 3344-3349) and in atrophying skeletal muscle.
The first step in degradation of many proteins involves their conjugation to Ub by an ATP-requiring process, as described below. The ubiquitinated proteins are then degraded by an ATP-dependent proteolytic complex, referred to above, known as the 26S proteasome complex.
The precise nature of the 26S proteasome complex is unclear, although it has been shown that the 1000-1500 kDa (26S) complex can be formed in extracts of energy-depleted reticulocytes by an ATP-dependent association of three components, referred to as CF-1, CF-2, and CF-3 (Ganoth et al., 1988,
J. Biol. Chem.,
263: 12412-12419). A large (~700 kDa) multimeric protease found in the cytoplasm and nucleus of eukaryotic cells, referred to as the proteasome, is a component (CF-3) (Driscoll et al., 1992,
J. Biol. Chem.,
265: 4789-4792; Eytan et al., 1989,
Proc. Natl. Acad. Sci. U.S.A.,
86: 7751-7755; Orlowski et al., 1990,
Biochemistry,
29: 10289-10297; Rivet, 1989,
Arch. Biochem. Biophys.,
268: 1-8).
The proteasome is believed to make up the catalytic core of the large 26S multisubunit cytoplasmic particle necessary for the ubiquitin-dependent pathway of intracellular proteolysis (Driscoll et al., 1990,
J. Biol. Chem.,
265: 4789-4692; Eytan et al., 1989,
Proc. Natl. Acad. Sci. U.S.A.,
86: 7751-7755; Hough et al., 1987,
Biochemistry,
262: 8303-8313; McGuire et al., 1988,
Biochim. Biophys. Acta.,
967: 195-203; Rechsteiner et al., 1987,
Ann. Rev. Cell. Biol.,
3: 1-30; Waxman et al., 1987,
J. Biol. Chem.,
262: 2451-2457). By itself, the proteasome is unable to degrade ubiquitinated proteins, but provides most of the proteolytic activity of the 26S proteasome complex.
There is another ATP-dependent protease that is involved in degradation of ubiquitinated proteins, forms a complex with the proteasome and appears to be part of the 26S proteasome complex, which rapidly degrades proteins conjugated to ubiquitin. This protease, referred to as multipain, has been identified in muscle and plays an essential role in the ATP/ubiquity-independent pathway.
The complex formed between multipain and proteasome in vitro appears very similar or identical to the 1500 kDa Ub-conjugate, degrading enzyme, or 26S proteolytic complex, isolated from reticulocytes and muscle. The complexes contain the characteristic 20-30 kDa proteasome subunits, plus a number of larger subunits, including the six large polypeptides found in multipain. The complex formed contains at least 10-12 polypeptides of 40-150 kDa. A 40 kDa polypeptide regulator of the proteasome, which inhibits the proteasome's proteolytic activities has been purified from reticulocytes and shown to be an ATP-binding protein whose release appears to activate proteolysis. The isolated regulator exists as a 250 kDa multimer and is quite labile (at 42° C.). It can be stabilized by the addition of ATP or a nonhydrolyzable ATP analog, although the purified regulator does not require ATP to inhibit proteasome function and lacks ATPase activity. The regulator has been shown to correspond to an essential component of the 1500 kDa proteolytic complex. The regulator appears identical to CF-2 by many criteria. These findings suggest that the regulator plays a role in the ATP-dependent mechanism of the 26S proteasome complex.
The 20S proteasome is composed of about 15 distinct 20-30 kDa subunits. It contains at least three different peptidases that cleave specifically an the carboxyl side of the hydrophobic, basic, and acidic amino acids (Goldberg et al., 1992,
Nature,
357: 375-379: Goldberg, 1992,
Eur. J. Biochem.,
203: 9-23; Orlowski, 1990,
Biochemistry,
29: 10289-10297; Rivett et al., 1989,
Arch. Biochem. Biophys.,
218: 1; Rivett et al., 1989,
J. Biol. Chem.,
264: 12215-12219; Tanaka et al., 1992,
New Biol.
4: 1-11). These peptidases are referred to as the chymotrypsin-like peptidase, the trypsin-like peptidase, and the peptidylglutamyl peptidase. Which subunits are responsible for these activities is unknown although the cDNA's encoding several subunits have been cloned (Tanaka et al., 1992,
New Biol.,
4: 1-11).
B. Ubiquitination and Phosphorylation in Protein Processing
As reviewed by Hopkin (1997,
J. NIH Research,
9: 36-42) and briefly summarized herein, insight into the mechanisms by which proteolysis is controlled come from studies of the eukaryotic cell cycle. To proceed through the cell cycle, replicating its genome and dividing the resulting DNA between daughter cells during mitosis, a cell must appropriately activate and inactivate the regulators of cell division, the cyclin-dependent kinases (Cdks). To control Cdks, cells can specifically degrade the cyclin proteins that activate Cdks and the inhibitors that inactivate them. One mechanism by which specificity in targeted proteolysis is achieved is ubiquitination, the process by which cells tack long chains of a 76-amino acid marker protein called ubiquitin (Ub) onto proteins that are destined for destruction. Ubiquitination of a handful of cyclins and Cdk inhibitors leads to their timely demise and allows a cell to complete mitosis or to replicate its DNA; further, it is believed that phosphorylation of unstable proteins, such as the cyclins, often increases their susceptibility to ubiquitination and subsequent elimination.
As described below, ubiquitination affects signal transduction, as it may mark certain cell-surface growth-factor receptors for endocytosis and destruction; further, it is known that ubiquitination, coupled with phosphorylation, stimulates the signaling pathway that activates the transcription factor NF&kgr;B. Ubiquitin also plays a role in protein degradation pathways regulating cell differentiation and death during development.
i. Ubiquitination and the Cell Cycle
Evidence that ubiquitination was interesting from the point of view of regulation came with the development of a mouse cell line that arrests in the G
2
, or gap 2, phase of the cell cycle; these cells harbor a defect that cripples an enzyme that activates Ub before it can bind to proteins, such as the cyclins, that must be targeted for destruction. Prior to this work, ubiquitination was viewed only as a means for eliminating damaged, denatured, and misfolded proteins.
Most of the proteolysis that occurs in cells involves the degradation of Ub-conjugated proteins. As stated above, the proteasome recognizes the polyubiquitin tag, selectively admits proteins to which this marker is complexed and then cleaves them into small peptide fragments. Ubiquitination is dependent upon a series of proteins named for their order of elution from a Ub-affinity column. Ub-activating enzymes, called E1s, prime Ub for transfer to a substrate protein by forming a temporary thioester linkage between a terminal glycine of Ub and one of their own cysteine residues. Enter the Ub-conjugating proteins generically called E2

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