Determining Cleavage Site Sequences to Characterize the Active Site of BACE1 Aspartic Protease
2015-04
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Determining Cleavage Site Sequences to Characterize the Active Site of BACE1 Aspartic Protease
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2015-04
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Proteases play key roles in physiology and disease development due to their active role in the regulation of other proteins. Understanding and characterizing the active site of relevant proteases provides information vital to the production of inhibitors and elucidates potential native substrates that may be affected by inhibitors. Once the preferred sequence of the active site of a target protease, such as β-secretase (BACE1), has been defined, protease inhibitors can be created to treat or manage diseases such as Alzheimer's disease (AD). BACE1 is an aspartic protease that is overexpressed in the AD brain and is believed to initiate the AD disease pathway. As such, it is a strong candidate for drug design. However, chronic administration of BACE1 inhibitors could result in undesirable side effects due to the impairment of its ability to hydrolyze native substrates; therefore, the amino acid peptide sequence preferentially cleaved by BACE1 needs to be characterized. Not only will this indicate potential substrates that may be affected by BACE1 inhibition, it will also aid in the synthesis of viable inhibitors. Recently, a novel method for determing the cleavage sequence of proteases was reported. Proteomic identification of cleavage sites (PICS) is a method designed to accurately identify cleavage sequence preferences and neighbor interactions in the cleavage site that influence protease cleavage. This method gives additional information with less bias than previous methods used to characterize protease active sites. Multiple controls were used to confirm the validity of the procedure. These controls demonstrated our ability to successfully identify the amino acid sequence preferences for well characterized proteases. We were thus able to confidently use PICS to obtain the sequence of amino acids preferentially cleaved by BACE. BACE1A has two noticeable characteristics for the amino acid sequence cleaved: aromatic amino acids are preferred in the P1 site and leucine is strongly preferred in P2'. Other preferred amino acids are observed in the sequence, but not to the extent of P1 and P2'. Neighbor interactions were also investigated. Positive cooperativity resulted with leucine or valine in P3 and with phenylalanine or tyrosine in P1. There were strong interactions between valine in P3 and phenylalanine in P1 and between tyrosine in P1 and valine in P2'. Sequence preferences were also investigated for BACE2A, which exhibited both similarities and differences between BACE1A and BACE2A. The next step in this research will be to use knowledge of the preferred cleavage sites to determine physiological substrates of BACE1A. This will reveal more information about the natural function of BACE1A and identify potential side effects of its inhibition.
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University of Minnesota M.S. thesis. April 2015. Major: Chemistry. Advisor: Joseph Johnson. 1 computer file (PDF); xi, 89 pages.
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Heink, Nicole. (2015). Determining Cleavage Site Sequences to Characterize the Active Site of BACE1 Aspartic Protease. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/174780.
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