18036, a novel calpain-like protease and uses thereof

Details 18036, a novel calpain-like protease and uses thereof 18036, a novel calpain-like protease and uses thereof

2001-02-26

2003-09-16

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S219000, C435S069100, C435S325000, C435S320100, C435S252300, C536S023200

Reexamination Certificate

active

06620592

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to novel calpain-like protease nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
BACKGROUND OF THE INVENTION
Calpains refer to calcium-activated neutral proteinases, a superfamily of endopeptidases typically having cysteine-proteinase and calcium-binding characteristics. These proteinases cleave numerous substrate proteins in a limited manner, typically leading to modification of the function and/or activity rather than general degradation of the substrate.
Calpains are classified into two main groups, the typical or conventional calpains and the atypical calpains, based on their domain content and/or variation. The typical calpains are further subdivided into ubiquitous and tissue-specific calpains based on their predominate patterns of expression.
Two forms of ubiquitous calpains have been extensively characterized in vertebrates: the &mgr;-calpains (calpain I, CAPN1) and the m-calpains (calpain II, CAPN2), which are activated in vitro by micro- and millimolar calcium concentrations, respectively. An intermediate &mgr;/m calpain has been characterized in chicken.
The ubiquitous &mgr;- and m-calpains are heterodimers, each having a distinct, but homologous, large 80 kDa subunit (referred to as &mgr;CL or mCL, respectively) and an identical small 30 kDa subunit (referred to as 30K or Cs). The large subunit has four domains, designated I-IV from the N-terminus to the C-terminus. The function of domain I is unclear. Domain II is the cysteine protease domain responsible for calpain protease activity. Domain III is homologous to a calmodulin-binding protein and is speculated to interact with the calcium-binding domains of the large (domain IV) and small subunits (domain VI), when calcium is bound, thereby freeing the protease domain for activity (Goll et al. (1992)
BioEssays
14:549-556). Domain IV of the large subunit is a calmodulin-like calcium-binding domain containing four EF-hand calcium-binding motifs. Although structurally similar to calmodulin, domain IV is more similar to sorcin, ALG-2, and grancalcin. Sorcin is involved in the multi-drug resistance of cultured cell lines and was recently reported to associate with the cardiac ryanodine receptor. Grancalcin possibly plays a role in granule-membrane fusion and degranulation. ALG-2 is thought to be involved in apoptosis and is induced by tumor promoters. See Meyers et al. (1995)
J. Biol. Chem.
270:26411-26418; Meyers et al. (1985)
J. Cell Biol.
100:588-597, Vito et al. (1996)
Science
271:521-525; Teahan et al. (1992)
Biochem. J.
286:549-554; Boyhan et al. (1992)
J. Biol. Chem.
267:2928-2933.
The large subunit of calpains is the catalytic subunit. Three non-contiguous amino acid residues, Cys, His, and Asn, residing within domain II are part of the active site. A recombinant calpain consisting essentially of domains I, II, and III showed calcium-independent activity. Thus, it has been concluded that domain II, but not IV, is necessary and sufficient for protease activity. See Vilei et al. (1997)
J. Biol. Chem.
272:25802-25808; and Suzuki et al. (1998)
FEBS Letters
433(1, 2):1-4.
The small subunit of typical calpains contains two domains, which are designated V and VI from the N-terminus to the C-terminus. Domain V is an N-terminal glycine-clustering hydrophobic region. Domain VI, which is similar to domain IV of the large subunit, is also a calcium-binding domain containing six EF-hands, EF2-EF5 as in the large subunit, and EF1 and EF6. EF5 of domain VI does not bind calcium and is proposed to be involved in the heterodimeric binding of domains IV and VI during interaction between the large and small subunits.
Not all calpains contain a small subunit, which is identified as a regulator of calpain activity by acting as an inhibitor or pseudosubstrate. In heterodimeric calpains, the small subunit may regulate the calcium-sensitivity of calpain by association and dissociation (Yoshizawa et al. (1995)
Biochem. Biophys. Res. Commun.
208:376-383). However, the subunits remain associated during catalysis (Zhang et at. (1996)
Biochem. Biophys. Res. Commun.
227:890-896).
The mechanism of activation of calpains is not entirely clear. Suggested mechanisms include combinations of N-terminal autolysis of subunits, homo- and heterodimer association/dissociation, the ratio and binding status of calpains to the calpain endogenous inhibitor calpastatin, calcium presence and concentration, and the redox state of the active site. See Johnson et al. (1997)
BioEssays
19(11):1011-1018.
Because &mgr;- and m-calpain are activated by in vitro calcium concentrations significantly above physiological levels, in vivo mechanisms that lower the calcium requirement have been proposed. Such mechanisms include interactions with membrane phospholipids and/or membrane associated proteins. See Inomata et al. (1990)
Biochem. Biophys. Res. Comm.
171:625-632; and Inomata et al. (1995)
Biochim. Biophys. Acta.
1235:107-114.
An activator protein specific for rat brain &mgr;-calpain has been isolated and sequenced by Melloni et al. (1998)
J. Biol. Chem.
273:12827-12831. Another activator protein specific for m-calpain is found in skeletal muscle. In addition, phospholipids, especially acidic phospholipids, have been found to greatly reduce the calcium concentration necessary for activation. Other activators and factors including DNA have been reported (Mellgren (1991)
J. Biol. Chem.
266:13920-13924).
Calpastatin is an endogenous inhibitor of most calpains, the tissue-specific calpain p94 being an exception. Calpastatin, which has five domains, is cleaved by calpain in the interdomain regions, generating inhibitory peptides. The inhibitory effect of calpastatin has been attributed to interactions with calpain domains II, III, IV, and VI. The reactive site of calpastatin shows no apparent homology to that of other protease inhibitors, and it contains the consensus sequence TIPPXYR (SEQ ID NO:6), which is essential for inhibition. See Kawasaki et al. (1989)
J. Biochem.
106:274-281, Croall et al. (1994)
Biochem.
33:13223-13230; Croall et al. (1991)
Physiol. Rev.
71:813-847; Kawasaki et al. (1996)
Mol. Membr. Biol.
13:217-224; Melloni et al. (1989)
Trends Neurosci.
12:438-444; Sorimachi et al. (1997)
J. Biochem.
328:721-732; and Johnson et al. (1997)
BioEssays
19(11):1011-1018.
Synthetic active-site inhibitors with varying specificities for calpain and other cysteine proteases include E-64 and derivatives of E-64; leupeptin (N-acetyl-Leu-Leu-argininal); calpain inhibitors I (N-acetyl-Leu-Leu-norleucinal) and II (N-acetyl-Leu-Leu-methioninal); oxoamide inhibitor molecules AK295, AK275, and CX275; and derivatives of peptidyl &agr;-oxo compounds. In contrast to these active-site inhibitors, PD150606 inhibits calpains by binding the calcium-binding domains. The combination of PD150606 and an active site inhibitor such as AK295 can inhibit calpain with high specificity. See Figueiredo-Pereira et al. (1994)
J. Neuro. Chem.
62:1989-1994); Tsubuki et al. (1996)
J. Biochem. (Tokyo)
119:572-576); and Sorimachi et al. (1997)
J. Biochem.
328:721-732. Wang et al (1997)
Advances in Pharmacology,
Volume 37.
Several typical tissue-specific calpains are known in vertebrates, including skeletal muscle p94 (nCL-1, calpain 3′, CAPN3), stomach nCL2 (CAPN4) and nCL 2′, and digestive tubule nCL4. While p94 contains EF hands, it does not require calcium for proteinase activity. p94 has a domain IV sequence similar to that of &mgr;CL and mCL, but it does not bind to a small 30 kDa subunit (Kinbara et al. (1997)
Arch. Biochem. Biophys.
342:99-107). p94 contains unique insertion sequences called IS1 and IS2, which are found in domain II and between domains III and IV, respectively). IS2 contains a nuclear-localization-signal-like basic sequence (Arg-Pro-Xaa-Lys-Lys-Lys-Lys-x-Lys-Pro). Connectin/titin binding is also attributed to IS2. p94 may change its localization in a cell-cycle dependent manner

Affiliated with

Also associated with

Rating

18036, a novel calpain-like protease and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with 18036, a novel calpain-like protease and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and 18036, a novel calpain-like protease and uses thereof will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3025495