Polyglutamine repeat sequences

Abstract

Several neurodegenerative diseases result from the aggregation of polyglutamine repeat proteins into insoluble neuronal intranuclear inclusions. The invention provides methods with which to study the processes of these diseases, including methods for solubilizing polypeptides containing a polyglutamine repeat sequence, for storing these polyglutamine polypeptides and inhibit their spontaneous aggregation, for making the aggregates of polyglutamine polypeptides, for assaying the extension of existing polyglutamine aggregates, for determining the ability of a chemical compound to inhibit aggregation, and for inhibiting aggregation of polyglutamine polypeptides. The invention further provides materials with which to study these diseases including a synthetic aggregate that have a capability to recruit additional monomeric polyglutamine polypeptides and chemical compounds that inhibit the formation and/or extension of polyglutamine aggregates.

1 . A method for making an aggregate of aggregation-prone polypeptides comprising obtaining a solution of the aggregation-prone polypeptides, freezing the solution containing the polypeptides, incubating the frozen polypeptides in a frozen state, and permitting the aggregates to form.  
   
   
       2 . The method of  claim 1  wherein the aggregation-prone polypeptide contains a polyglutamine repeat sequence.  
   
   
       3 . The method of  claim 2  wherein the solution comprises polypeptides comprising a polyglutamine repeat sequence of at least Q 35 .  
   
   
       4 . The method of  claim 1  wherein the freezing is a snap freezing.  
   
   
       5 . The method of  claim 1  which further comprises sonicating the aggregates.  
   
   
       6 . The method of  claim 5  which comprises, after the sonicating, filtering the aggregates.  
   
   
       7 . The method of  claim 6  wherein the filtration is through a membrane filter.  
   
   
       8 . The method of  claim 7  wherein the membrane filter is a 1.2 μm membrane filter.  
   
   
       9 . An in vitro produced aggregate that is made by the method of  claim 1 .  
   
   
       10 . An in vitro produced aggregate that is made by the method of  claim 5 .  
   
   
       11 . An in vitro produced aggregate that is made by the method of  claim 6 .  
   
   
       12 . A method for dissolving or disaggregating a polypeptide comprising combining the polypeptide in a mixture of trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP) and permitting the polypeptide to dissolve or disaggregate in the mixture.  
   
   
       13 . The method of  claim 12  wherein the polypeptide is a polyglutamine repeat polypeptide.  
   
   
       14 . The method of  claim 13  wherein the polyglutamine repeat polypeptide has a glutamine repeat sequence of Q 35  or more.  
   
   
       15 . The method of  claim 12  which further comprises removing the TFA and HFIP and resolubilizing the polypeptide in water.  
   
   
       16 . The method of  claim 12  wherein the ratio of TFA and HFIP in the mixture is between 20:1 and 1:20.  
   
   
       17 . The method of  claim 16  wherein the ratio is between 5:1 and 1:5.  
   
   
       18 . The method of  claim 17  wherein the ratio is about 1:1.