Human caspase-12 materials and methods

Details Human caspase-12 materials and methods Human caspase-12 materials and methods

2001-05-08

2004-12-21

Hutson, Richard (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S226000, C435S183000, C435S219000, C435S212000, C435S069100, C530S350000

Reexamination Certificate

active

06833248

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to novel caspase polynucleotides and polypeptides.
DESCRIPTION OF RELATED ART
Cysteine-dependent aspartate-specific proteases (caspases) are a family of proteases that cleave their substrates at aspartic acid (D)-X bonds. They are highly specific endopeptidases that catalyze limited proteolysis [Stennicke et al.,
Cell Death Differen.
(1999) 6:1054-1059]. To date 14 mammalian caspases have been identified. Caspase-2, -3, -6, -7, -8, -9 and -10 are major players in the execution phase of apoptosis, whereas caspase-1, -4, -5, and -11 are involved in cytokine processing. The function of caspase-13, and -14 have not been determined, although based on structure caspase-13 is more similar to those caspases active in cytokine processing whereas caspase-14 appears to be closely related to caspases associated with cell death [Slee et al.,
Cell Death Differen.
(1999) 6:1067-1074].
Caspases are initially expressed within cells as zymogens with low or no detectable enzymatic activity. The general primary structure of an unprocessed caspase consists of an amino-terminal prodomain, followed by a large subunit and a small subunit. Proteolytic processing of the zymogen either in trans or cis results in one or more cleavages at specific aspartic acid residues present between the large and small subunits. In most caspases, cleavage also occurs between the prodomain and the large subunit. The mature active caspase is a heterotetramer, which consists of two large (~20 kDa) and two small (~10 kDa) subunits. Residues from both subunits contribute to the two substrate-binding pockets which recognize at least four amino acids (P
1
, P
2
, P
3
, and P
4
) located N-terminal to the cleavage site in substrates. A highly conserved pentapeptide (QACXG) containing the catalytic cysteine, present in the large subunit, leads to the absolute requirement for an Asp at the P
1
position. Residues found at positions P
2
, P
3
and P
4
on substrates vary greatly and determine substrate specificity within the caspase family [Stennicke et al.,
Cell Death Differen.
(1999) 6:1054-1059; Takahashi,
Int J Hematology
(1999) 70:226-232; Slee et al.,
Cell Death Differen.
(1999) 6:1067-1074].
Caspases can be divided into two groups based on the length of their N-terminal prodomains. Caspases with long prodomains such as caspase-1, -2, -4, -8, -9, and -10 contain regions such as caspase-recruiting domains (CARD) or death effector domains (DED), that mediate interactions with other proteins. These interactions cause oligomerization of the caspases which is thought to trigger autoprocessing of these proteins. Once activated, these caspases cleave downstream caspases which contain short prodomains and are incapable of autocatalysis, creating a proteolytic cascade [Slee et al.,
Cell Death Differen.
(1999) 6:1067-1074; Kumar,
Cell Death Differen.
(1999) 6:1060-1066]].
Initial expression as relatively inert zymogens is especially important since most caspases are expressed constitutively. Regulation of caspase activity may also occur by controlling the subcellular localization of caspases. For example, most caspases are present in the cytosol, however, a number of their substrates are localized in organelles such as the nucleus. Catalytically inactive isoforms of caspases, e.g., caspase-9, which are generated by alternative splicing, can interfere with caspase activation thereby inhibiting apoptosis. Some caspases, e.g., caspase-9 can be regulated by post-translational modifications: phosphorylation inhibits its processing and activation. Negative regulators of caspases called inhibitors of apoptosis (IAPs) have also been identified. It is thought that IAPs protect from cell death by interfering with the catalytic activity of caspases or by preventing the processing and activation of caspases [Takahashi,
Int J Hematology
(1999) 70:226-232; Slee et al.,
Cell Death Differen.
(1999) 6:1067-1074; Kumar,
Cell Death Differen.
(1999) 6:1060-1066].
Currently, two distinct pathways have been identified that lead to caspase activation in apoptosis: the cell surface death receptor pathway and the mitochondria-initiated pathway. The cell surface death receptor pathway is initiated by ligation of cell surface death receptors, e.g., Fas and tumor necrosis factor receptor 1 (TNFR1), leading to ligand-induced receptor trimerization, recruitment of intracellular receptor-associated proteins as well as caspases with long prodomains, and ultimately to the activation of caspases. The mitochondria-initiated pathway begins with the release of cytochrome c from the mitochondria in response to various stimuli, e.g., DNA damage. Cytosolic cytochrome c binds to apoptotic protease activating factor 1 (Apaf1) forming an Apaf1-cytochrome c multimeric complex, which then associates with caspase-9 thereby triggering the activation of several downstream caspases. Both pathways lead to enzymatically active caspases that cleave substrates including poly (ADP-ribose) polymerase (PARP), PKC&dgr;, and cPLA
2
[Takahashi,
Int J Hematology
(1999) 70:226-232; Slee et al.,
Cell Death Differen.
(1999) 6:1067-1074; Kumar,
Cell Death Differen.
(1999) 6:1060-1066].
Caspases have also been shown to be important for the processing of the cytokines interleukin-1&bgr; (IL-1&bgr;) and interleukin-18 (IL-18). Both IL-1&bgr; and IL-18 are expressed as inactive precursor proteins that are proteolytically processed by caspases to yield active cytokines involved in inflammatory responses. IL-1&bgr; is a multifunctional protein present in a number of different cell types. For example, IL-1&bgr; is a growth factor for acute myeloid leukemia cells, is produced by murine monocytes that migrate into Peyer's patches during inflammation, and is associated with prolonged longevity of peripheral blood monocytes in vitro. IL-18 is a crucial cytokine involved in IFN-&ggr; production in Th1 cells and natural killer cells during inflammation [Zeuner et al.,
Cell Death Differen.
(1999) 6:1075-1080].
Recent studies have also uncovered possible physiological roles for caspases apart from apoptosis. Pro-apoptotic caspase-3 is suggested to be involved in processes such as T cell proliferation, IL-2 release in PHA-stimulated Jurkat T cells, IL-16 processing in CD8
+
and CD4
+
T cells, and in cell cycle control [Zeuner et al.,
Cell Death Differen.
(1999) 6:1075-1080]. Caspases have also been implicated in regulating terminal lens fiber differentiation. The mature lens fibers do not undergo cell death, however, they do exhibit nuclear degeneration similar to that seem in apoptosis. Finally, caspases have been shown to mediate CD95 inhibition of erythroid differentiation by cleaving specific transcription factors [Zeuner et al.,
Cell Death Differen.
(1999) 6:1075-1080]. Although the most extensively examined process associated with caspase is apoptosis, they appear to function in several other processes.
Of interest to the present invention is Van de Craen et al.,
FEBS Lett.
(1997) 403:61-69, which describes the identification and cloning of murine caspase-12. Murine caspase-12 is predominantly expressed in skeletal muscle and lung, and moderately expressed in brain, heart, spleen, liver, kidney and testis. Northen analysis indicates the presence of several different size murine caspase-12 transcripts suggesting that different isoforms of murine caspase-12 may exist. Transient transfection of murine caspase-12 into HeLa and Rat1 cells resulted in the induction of apoptosis [Van de Craen et al.,
FEBS Lett.
(1997) 403:61-69].
Nakagawa et al., Nature (2000) 403: 98-103, suggests a potential role for mouse caspase-12 in neurodegenerative disease. Nakagawa et al. reported that cortical neurons isolated from caspase-12 null mutant mice were relatively resistant to induction of apoptosis by &bgr;-amyloid. Compared to neurons from wild-type animals, neurons from the caspase-12 null mice could be

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