Sirt4 activities

Abstract

It has been discovered that Sirt4 possesses an ADP-ribosyltransferase activity. Sirt4 is localized to mitochondria, where it binds to and regulates the activity of proteins such as glutamate dehydrogenase. The ADP-ribosyltransferase activity of Sirt4 is important for the regulation of biological functions such as insulin secretion. Methods of screening for compounds that modulate the expression or activity of Sirt4 are provided. Also provided are methods of modulating insulin secretion, treating metabolic disorders, and treating neurodegenerative disorders by modulating the expression or activity of Sirt4.

1 . A method of evaluating Sirt4 activity, the method comprising: 
 providing a cell-free composition that comprises a Sirt4 protein, an ADP-ribosyl donor, and a substrate; and    evaluating ADP-ribosylation activity in the composition.    
     
     
         2 . The method of  claim 1 , wherein the ADP-ribosyl donor comprises NAD.  
     
     
         3 . The method of  claim 2 , wherein the NAD is radio-labeled.  
     
     
         4 . The method of  claim 1 , wherein the substrate comprises a peptide.  
     
     
         5 . The method of  claim 1 , wherein the substrate comprises GDH, aldehyde dehydrogenase, or histones, or a fragment of any thereof.  
     
     
         6 . The method of  claim 1 , wherein the providing comprises combining a preparation of the Sirt4 protein that is at least 10% pure with the ADP-ribosyl donor and the substrate.  
     
     
         7 . The method of  claim 1 , wherein the Sirt4 protein comprises a sequence at least 85% identical to SEQ ID NO:3.  
     
     
         8 . The method of  claim 1 , wherein the Sirt4 protein comprises a sequence at least 85% identical to SEQ ID NO:1 or amino acid residues 29-314, 29-308, 30-314, 36-308, 42-308, 42-300, or 56-314 of SEQ ID NO:1.  
     
     
         9 . The method of  claim 1 , further comprising including a test compound in the cell-free composition.  
     
     
         10 . A method of evaluating the effect of a test compound on Sirt4, the method comprising: 
 a) providing a reaction mixture comprising Sirt4 and a test compound; and    b) evaluating an activity of Sirt4.    
     
     
         11 . The method of  claim 10 , wherein the activity is ADP-ribosyltransferase activity.  
     
     
         12 . The method of  claim 10 , wherein the reaction mixture comprises NAD or an NAD analog.  
     
     
         13 . The method of  claim 10 , wherein the NAD or NAD analog comprises a radioactive label.  
     
     
         14 . The method of  claim 10 , wherein the reaction mixture comprises an ADP-ribosylation substrate selected from the group consisting of: GDH, aldehyde dehydrogenase, and histones.  
     
     
         15 . The method of  claim 10 , wherein the test compound is a small molecule.  
     
     
         16 . The method of  claim 10 , wherein the method is repeated for each of a plurality of test compounds from a chemical library.  
     
     
         17 . The method of  claim 10 , wherein the Sirt4 comprises a sequence at least 85% identical to SEQ ID NO:3.  
     
     
         18 . The method of  claim 10 , wherein the Sirt4 comprises a sequence at least 85% identical to SEQ ID NO:1 or amino acid residues 29-314, 29-308, 30-314, 36-308, 42-308, 42-300, or 56-314 of SEQ ID NO: 1.  
     
     
         19 . The method of  claim 10 , further comprising c) comparing the activity evaluated in (b) with the Sirt4 activity evaluated in the absence of the test compound.  
     
     
         20 . A method of identifying a compound that alters a Sirt4-associated parameter in a cell, the method comprising: 
 a) contacting a test compound to a cell that expresses Sirt4; and    b) evaluating a Sirt4-associated parameter associated with the cell.    
     
     
         21 . The method of  claim 20 , wherein the Sirt4-associated parameter is ADP-ribosylation activity.  
     
     
         22 . The method of  claim 20 , wherein the test compound is a small molecule.  
     
     
         23 . The method of  claim 20 , wherein the method is repeated for each of a plurality of test compounds from a chemical library.  
     
     
         24 . The method of  claim 20 , further comprising 
 c) comparing the parameter determined in (b) with a Sirt4-associated parameter evaluated in the absence of the test compound.    
     
     
         25 . A method of modulating insulin secretion in response to glucose, the method comprising modulating the expression or activity of Sirt4 in an insulin-secreting cell.  
     
     
         26 . The method of  claim 25 , wherein insulin secretion is increased by decreasing the expression or activity of Sirt4.  
     
     
         27 . The method of  claim 25 , wherein insulin secretion is decreased by increasing the expression or activity of Sirt4.  
     
     
         28 . The method of  claim 25 , wherein insulin secretion is modulated in vitro.  
     
     
         29 . The method of  claim 25 , wherein insulin secretion is modulated in a subject.  
     
     
         30 . The method of  claim 25 , wherein the modulation of Sirt4 expression or activity is effected by administering an agent that modulates Sirt4 expression or activity.  
     
     
         31 . A method of treating or preventing a metabolic disorder, the method comprising administering, to a subject, an agent that modulates the expression or activity of Sirt4, in an amount effective to treat or prevent the metabolic disorder.  
     
     
         32 . The method of  claim 31 , wherein the metabolic disorder is diabetes, insulin resistance, metabolic syndrome, or pre-diabetes.  
     
     
         33 . The method of  claim 31 , wherein the levels of Sirt4 are modulated in an insulin-secreting cell.  
     
     
         34 . The method of  claim 31 , wherein the agent is an antagonistic nucleic acid that reduces Sirt4 expression.  
     
     
         35 . The method of  claim 31 , wherein the agent comprises an siRNA that targets Sirt4 mRNA.  
     
     
         36 . A method of treating or preventing a symptom of a neurodegenerative disorder, the method comprising administering to a subject a compound that increases the expression or activity of Sirt4 in an amount effective to treat or prevent the neurodegenerative disorder.  
     
     
         37 . The method of  claim 36 , wherein the neurodegenerative disorder involves accumulation of β-amyloid peptide.  
     
     
         38 . The method of  claim 36 , wherein the neurodegenerative disorder is Alzheimer's disease.