Promotion of peroxisomal catalase function in cells
The molecular mechanisms of peroxisome biogenesis have begun to emerge: in contrast, relatively little is known about how the organelle functions as cells age. The present inventors characterized age-related changes in peroxisomes of human cells and showed that aging compromises peroxisomal targeting signal 1 (PTS 1) protein import, with the critical antioxidant enzyme, catalase, especially affected. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite, H 2 O 2 , and this increased load of reactive oxygen species (ROS) may further reduce peroxisomal protein import and exacerbate the effects of aging. Disclosed are novel compositions and methods for restoring catalase in peroxisomes by use of targeted catalase modified at its C-terminus and/or N-terminus, optionally in combination with polypeptides which promote cellular uptake of proteins, to prevent or overcome the changes that follows aging or that are associated with a number of diseases or disorders.
1. A modified human catalase polypeptide having a carboxy-terminal peroxisome targeting signal (PTS) that has been modified from a native sequence of Lys-Ala-Asn-Leu (SEQ ID NO: 1) by replacement of SEQ ID NO: 1 in human catalase with a PTS comprising the sequence Xaa −3 Xaa −2 Xaa −1 , wherein, independently,
Xaa −3 is Ser, Ala or Cys;
Xaa −2 is Lys, Arg or His; and
Xaa −1 Leu or Met, and
wherein the replacement sequence comprises, to the amino-terminal side of Xaa −3 , n additional amino acid residues wherein n is an integer between 2 and about 17, the additional residues being numbered sequentially from Xaa −4 for the first additional residue to Xaa −20 for the seventeenth additional residue.
2. The modified catalase polypeptide of claim 1 , wherein n is between about 5 and about 17.
3. The modified catalase polypeptide of claim 2 , wherein n is between about 7 and about 13.
4. The modified catalase polypeptide of claim 2 , wherein n is between about 5 and about 2.
5. The modified catalase polypeptide of claim 2 , wherein n is 9.
6. The modified catalase polypeptide of claim 1 , wherein residues at any one of Xaa −6 to Xaa −4 are hydrophobic amino acids.
7. The modified catalase polypeptide of claim 6 , wherein residues at any one of Xaa −8 to Xaa −4 are, independently, Leu, Val, lie, Ala or Gly.
8. The modified catalase polypeptide of claim 1 , wherein residue Xaa −4 is a positively charged amino acid.
9. The modified catalase polypeptide of claim 8 , wherein residue Xaa −4 is Lys, Arg or His.
10. The modified catalase polypeptide of claim 9 , wherein residue Xaa −4 is Lys.
11. The modified catalase polypeptide of claim 1 , wherein Xaa −3 is Ser, Xaa −2 is Lys, and Xaa −1 is Leu.
12. A modified catalase polypeptide according to claim 2 , wherein the four C-terminal amino acids are encoded by the coding nucleotides from a reverse primer, the sequence of which is SEQ ID NO:18.
13. The modified catalase polypeptide of claim 1 , wherein, when the polypeptide is contacted with cells in vitro, it is imported into peroxisomes at a rate that exceeds the rate of import of native human catalase under the same conditions.
14. A pharmaceutical composition comprising:
(a) the modified catalase polypeptide of claim 1 ; and
(b) a pharmaceutically acceptable excipient or carrier.
15. A pharmaceutical composition comprising:
(a) the modified catalase polypeptide of claim 2 ; and
(b) a pharmaceutically acceptable excipient or carrier.
16. A pharmaceutical composition comprising:
(a) the modified catalase polypeptide of claim 13 ; and
(b) a pharmaceutically acceptable excipient or carrier.
17. A deliverable, peroxisomally-targeted polypeptide comprising:
(a) the modified catalase polypeptide of claim 1 , and
(b) a delivery or translocation molecule or moiety bound thereto or associated therewith.
18. A deliverable, peroxisomally-targeted polypeptide comprising:
(a) the modified catalase polypeptide of claim 2 , and
(b) a delivery or translocation molecule or moiety bound thereto or associated therewith.
19. The deliverable, peroxisomally targeted polypeptide of claim 17 , wherein the delivery molecule is a peptide or polypeptide.
20. The deliverable polypeptide of claim 19 wherein the peptide or polypeptide is selected from the group consisting of
(a) HIV-TAT protein or a translocationally active derivative thereof,
(b) penetratin having the sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 4),
(c) a penetratin variant W48F having the sequence RQIKIFFQNRRMKWKK (SEQ ID NO: 5)
(d) a penetratin variant W56F having the sequence RQIKIWFQNRRMKFKK (SEQ ID NO: 6)
(e) a penetratin variant having the sequence RQIKIWFQNRRMKFKK (SEQ ID NO:7)
(f) herpes simplex virus protein VP22 or a translocationally-active homologue thereof from a different herpes virus; and
(g) Pep-1, having the sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO:9).
21. The deliverable polypeptide of claim 17 wherein the delivery moiety associated with the modified catalase is a liposome which comprises effective concentrations of external membrane phosphatidylserine for uptake by phagocytic cells or other phosphatidylserine-recognizing cells.
22. A method for reducing the concentration of hydrogen peroxide in a cell, comprising contacting said cell with a modified catalase polypeptide of claim 1 , under conditions wherein said polypeptide is targeted to peroxisomes in an amount sufficient to reduce said concentration.
23. The method of claim 22 , wherein the modified catalase polypeptide further comprises a delivery or translocation molecule or moiety bound thereto or associated therewith.
24. The method of claim 22 , wherein the contacting is in vitro.
25. The method of claim 22 , wherein the contacting is in vivo.
26. A method for treating a mammalian subject suffering from a disease or condition associated with or caused by an inadequate level of peroxisomally active catalase, comprising administering to the subject an effective amount of the modified catalase polypeptide of claim 1 .
27. A method for treating a subject suffering from a disease or condition associated with or caused by an inadequate level of peroxisomally active catalase, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 14 .
28. A method for treating a subject suffering from a disease or condition associated with or caused by an inadequate level of peroxisomally active catalase, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 15 .
29. The method of claim 26 , wherein the subject is a human.
30. The method of claim 26 , wherein the disease or condition is age-related.
31. The method of claim 26 wherein said administering is topical.