Muteins of the pyrroline-5-carboxylate reductase 1

Abstract

The invention relates to muteins of the pyrroline-5-carboxylate reductase 1 (PYCR1), to nucleic acid molecules comprising a nucleotide sequence encoding such muteins, to methods of determining in a subject a predisposition of having an age related disorder associated with PYCR1, to methods of identifying a compound capable of modifying the expression of PYCR1, and methods of treating a subject having an age-related disorder associated with PYCR1. The invention further relates to a genetically modified animal and a method of modifying the expression of the PYCR1 gene in an animal.

What is claimed is: 
     
         1 - 68 . (canceled) 
     
     
         69 . A method of identifying a compound capable of modifying the activity of a PYCR1 mutein, the method comprising:
 contacting a compound of interest with the mutein of pyrroline-5-carboxylate reductase 1 (PYCR1) or a nucleic acid molecule encoding a mutein of PYCR1, and   b. measuring the activity of the PYCR1 mutein,   whereby the PYCR1 mutein comprises or consists of an amino acid sequence wherein at least one of the amino acid residue corresponding to sequence positions 4, 119, 179, 189, 206, 251, 257 and 266 of the wild type amino acid sequence of PYCR1 as set forth in SEQ ID NO 4 is mutated and leads to a reduced function or loss of function of the mutein compared to the wild-type protein, wherein the amino acid residue at sequence position 251 is replaced by histidine.   
     
     
         70 . The method according to  claim 69 , wherein the amino acid residue at sequence position 257 is replaced by a hydrophilic amino acid. 
     
     
         71 . The method according to  claim 70 , wherein the hydrophilic amino acid is a hydroxyl-containing amino acid. 
     
     
         72 . The method according to  claim 70 , wherein the hydrophilic amino add is serine or threonine. 
     
     
         73 . The method according to  claim 69 , wherein the amino acid residue at sequence position 266 is replaced by glutamine or asparagine. 
     
     
         74 . A method of modifying the activity of a pyrroline-5-carboxylate reductase (PYCR1) mutein in an cell, the method comprising administering to the cell a compound capable of modifying the activity of the PYCR1 mutein. 
     
     
         75 . The method according to  claim 74 , wherein the compound comprises a nucleic add molecule that is capable of modifying the expression of the PYCR1 gene. 
     
     
         76 . The method according to  claim 75 , wherein the nucleic acid molecule is selected from the group consisting of antisense RNA, short interfering RNA (siRNA), micro RNA (miRNA), peptide nucleic acid (PNA) and combinations thereof. 
     
     
         77 . The method according to  claim 74 , wherein the amount of the PYCR1 mutein is increased. 
     
     
         78 . The method according to  claim 74 , wherein the amount of the PYCR1 mutein is inhibited. 
     
     
         79 . The method according to  claim 74 , wherein the cell is comprised in a mammal. 
     
     
         80 . The method according to  claim 79 , wherein the mammal is a human or a rodent. 
     
     
         81 . The method according to  claim 75 , wherein the nucleic acid molecule is operably linked to a regulatory sequence comprising a promoter sequence to allow expression of the nucleic acid molecule. 
     
     
         82 . The method according to  claim 75 , wherein the nucleic acid molecule is comprised in a vector. 
     
     
         83 . A nucleic acid molecule comprising a nucleotide sequence encoding a pyrroline-5-carboxylate reductase 1 (PYCR1) mutein comprising or consisting of an amino acid sequence wherein at least one of the amino acid residue corresponding to sequence positions 4, 119, 179, 189, 206, 251, 257 and 266 of the wild type amino acid sequence of PYCR1 as set forth in SEQ ID NO. 4. 
     
     
         84 . A method of determining in a subject a predisposition of having an age-related disorder associated with pyrroline-5-carboxylate reductase 1 (PYCR1), the method comprising analyzing a nucleic add sample obtained from a subject for the presence of a nucleotide sequence encoding a mutein comprising or consisting of an amino add sequence wherein at least one of the amino acid residue corresponding to sequence positions 4, 119, 179, 189, 206, 251, 257 and 266 of the wild type amino add sequence of PYCR1 as set forth in SEQ ID NO. 4, wherein the presence of the nucleotide sequence indicates a predisposition of the subject having or being at risk for having the age-related disorder. 
     
     
         85 . The method according to  claim 84 , wherein the age-related disorder is selected from the group consisting of cutis laxa (wrinkly skin syndrome), de Barsy syndrome and gerodermia osteodysplasia. 
     
     
         86 . A method of using a nucleic acid molecule comprising a nucleotide sequence encoding a mutein comprising or consisting of an amino acid sequence wherein at least one of the amino acid residue corresponding to sequence positions 4, 119, 179, 189, 206, 251, 257 and 266 of the wild type amino acid sequence of PYCR1 as set forth in SEQ ID NO. 4 for diagnosing or determining a predisposition of having an age-related disorder associated with PYCR1. 
     
     
         87 . The method according to  claim 86 , wherein the age-related disorder is selected from the group consisting of cutis laxa (wrinkly skin syndrome), de Barsy syndrome and gerodermia osteodysplasia. 
     
     
         88 . A method of treating a subject having an age-related disorder associated with PYCR1 or being at risk to develop an age-related disorder associated with PYCR1, the method comprising introducing a nucleic add molecule comprising a nucleotide sequence encoding pyrroline-5-carboxylate reductase 1 (PYCR1) or a functional fragment or functional mutant thereof into the subject. 
     
     
         89 . A method of treating a subject having an age-related disorder associated with PYCR1 or being at risk to develop an age-related disorder associated with PYCR1, the method comprising administering to the subject a compound capable of modifying the activity of a PYCR1 mutein.