Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1996-10-07
2001-04-10
Spector, Lorraine (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S300000, C530S350000
Reexamination Certificate
active
06214794
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the field of protein processing and protein signalling pathways and specifically to two novel proteins having distinct activities, which are derived from a common hedgehog protein precursor.
2. Description of the Related Art
Embryologists have long performed experimental manipulations that reveal the striking abilities of certain structures in vertebrate embryos to impose pattern upon surrounding tissues. Speculation on the mechanisms underlying these patterning effects usually centers on the secretion of signaling molecule that elicits an appropriate response from the tissues begin patterned. More recent work aimed at the identification of such signaling molecules implicates secreted proteins encoded by individual members of a small number of gene families. One such family of proteins which may have an influential effect upon patterning activities are those proteins encoded by the hedgehog gene family.
The hedgehog (hh) gene was initially identified based on its requirement for normal segmental patterning in Drosophila (Nüsslein-Volhard, C. & Wieschaus, E,
Nature
287:795-801, 1980). Its functions include local signaling to coordinate the identities of adjacent cells within early embryonic segments (Hooper, J. E., & Scott, M. P.
Early Embryonic Development of Animals
, pp.1-48, 1992) and a later function in cuticle patterning that extends across many cell diameters (Heernskerk, J. & DiNardo, S.,
Cell
, 76:449-460, 1994). The hh gene also functions in the patterning of imaginal precursors of adult structures, including the appendages and the eye (Mohler,
J. Genetics
, 120:1061-1072, 1988; Ma, et al.,
Cell
, 75:927-938, 1993; Heberlein, et al,
Cell
, 75:913-926, 1993; Tabata, T. & Kornberg, T. D.,
Cell
, 76:89-102, 1992; Basler, K. & Struhl, G.,
Nature
, 368:208-214,1994). Genetic and molecular evidence indicates that hedgehog proteins are secreted and function in extracellular signaling (Mohler, J., supra; Lee, et al,
Cell
, 71:33-50, 1992; Taylor, et al.,
Mech. Dev
, 42:89-96, 1993).
In vertebrates activities encoded by hh homologues have been implicated in anterior/posterior patterning of the limb (Riddle, et al.,
Cell
, 75:1401-1416, 1993; Chang, et al.,
Development
, 120:3339, 1994), and in dorsal/ventral patterning of the neural tube (Echelard, et al.,
Cell
, 75:1417-1430, 1993; Krauss, et al.,
Cell
, 75:1431-1444, 1993; Roelink, et al.,
Cell
, 76:761-775, 1994).
The vertebrate ventral midbrain contains neurons whose degeneration or abnormal function are linked to a number of diseases, including Parkinson's disease and schizophrenia. It is known that motor neurons develop in close proximity to the floor plate in the ventral midbrain. Midbrain projections to the striatum are involved in the control of voluntary movement (Bjorklund and Lindvall, In:
Handbook of Chemical Neuroanatomy
, eds., Borklund, et al., Amsterdam: Elsevier, pp55-122, 1984) and loss of these neurons results in the motor disorders of Parkinson's disease (Hirsch, et al.,
Nature
, 334:345, 1988). Midbrain dopaminergic neurons that innervate limbic structures and the cortex influence emotional and cognitive behavior, respectively, and abnormal function of these neurons has been associated with schizophrenia and drug addiction (Seeman, et al.,
Nature
, 365:441, 1993).
While the molecular nature of the factors that specify neuronal cell fate have not been established, members of the transforming growth factor-&bgr; (TGF-&bgr;) (Lyons, et al.,
Trends in Genetics
, 7:408, 1991) or the hedgehog protein family (Smith, J. C.,
Cell
, 76:193, 1994) may possess the characteristics expected from such factors as they participate in specification of cell fate, mediate inductive interactions between tissues, and in many cases act at a distance of only a few cell diameters.
The present invention establishes that hh activities encoded by these genes play a crucial role in early patterning of the developing eye and in patterning of the brain. For the first time, the invention shows that internal cleavage of hedgehog protein product is critical for full function, and that the two novel products of this auto-proteolytic cleavage display distinguishable activities, thus demonstrating that hh signaling activity is a composite effect of two separate signaling proteins that derive from a common hh protein precursor. In so doing, the invention provides the means for specific patterning and proliferation of desired neuronal cell types for addressing disorders which arise from neuronal degeneration or abnormal function.
SUMMARY OF THE INVENTION
The present invention is based on the seminal discovery that hedgehog proteins undergo auto-proteolytic cleavage which results in two separate proteins having distinct functional and structural characteristics. The two polypeptides, referred to as the “N” and “C” fragments of hedgehog, or N-terminal and C-terminal fragments, respectively, are produced after specific cleavage at a Gly↓Cys Phe site recognized by the autoproteolytic domain in the native protein. The “C” fragment functions as a cholesterol transferase during autoproteolysis thus allowing cholesterol modification of the “N” fragment.
Thus, in one embodiment, the invention provides a substantially pure polypeptide characterized by having an amino acid sequence derived from amino terminal amino acids of a hedgehog protein and having at its carboxy terminus, a Gly↓Cys Phe cleavage site specifically recognized by a proteolytic activity of the carboxy terminal fragment of the native hedgehog polypeptide.
In another embodiment, the invention provides a substantially pure polypeptide characterized by having an amino acid sequence derived from carboxy terminal amino acids of a hedgehog protein and having at its amino terminus, a Gly↓Cys Phe cleavage site specifically recognized by a proteolytic activity of the carboxy terminal fragment of the native hedgehog polypeptide.
The invention also provides a method for modulating proliferation or differentiation of neuronal cells, comprising contacting the cells with a hedgehog polypeptide. The native hedgehog polypeptide, the N, or the C fragment, or functional fragments derived therefrom, are most useful for the induction of proliferation or differentiation of neuronal cells substantially derived from floor plate neuronal cells.
In yet another embodiment, the invention provides a method for identifying a compound which affects hedgehog activity comprising incubating the compound with hedgehog polypeptide, or with biologically active fragments thereof, or with a recombinant cell expressing hedgehog, under conditions sufficient to allow the components to interact; and determining the effect of the compound on hedgehog activity or expression. For example, cholesterol level is measured as an indicator of hedgehog activity.
REFERENCES:
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patent: 5789543 (1998-08-01), Ingham et al.
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Ngo et al (1994) in: The Protein Folding Problem and Tertiary Structure Prediction. Merz et al. (eds) Birkhauser, Boston pp. 433, 492-495.*
Echelard et al. (1993) Cell 75:1417-1430.*
Placzck et al. (1993) Development 117:205-218.*
Hamerschnt et al. (1997) Trends Genet. 13:14-15.*
Lee et al., “Autoproteolysis in hedgehog Protein Biogenesis”,Science, 266:1528-1537, Dec. 2, 1994.
Tabata and Kornberg, “Hedgehog is a Signaling Protein with a Key Role in Patterning Drosphila Imaginal Discs”,Cell, 76:89-102, Jan. 14, 1994.
Krauss et al., “A Functionally Conserved Homolog of the Drosophila Segment Polarity Gene hh Is Expressed in Tissues with Polarizing Activity in Zebrafish Embryos”,Cell, 75:1431-1444, Dec. 31, 1993.
Ganong, William F., Review of Medical Physiology, Norwalk, Connecticut: Appleton and Lange, 1995, pp. 273 and 438.
Mohler et al., “Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in segmental patterning of Drosphila, ”Development, 1992, vol
Beachy Philip A.
Moon Randall T.
Porter Jeffrey A.
Gray Cary Ware & Freidenrich LLP
Haile, Ph.D. Lisa A.
Kaufman Claire M.
Spector Lorraine
The Johns Hopkins University School of Medicine
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