Abstract
Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the “a disintegrin and metalloprotease” (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ-secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs.
Highlights
In 2012, two proteomic studies were published which used primary neurons treated with a BACE inhibitor to identify proteins with reduced abundance in the secretome
The first substrates of sheddases and intramembrane proteases were largely identified by candidate approaches, partially driven by the phenotypes of the corresponding protease knock-out mice
One example is the loss-of-Notch-function phenotype which allowed to identify Notch as a substrate for ADAM10 and γ-secretase (De Strooper et al, 1999; Struhl and Greenwald, 1999; Hartmann et al, 2002). Another example is the hypomyelination of BACE1-deficient mice which led to the identification of neuregulin-1 as a BACE1 substrate (Hu et al, 2006; Willem et al, 2006)
Summary
FOR MASS SPECTROMETRY BASED SUBSTRATE IDENTIFICATION OF MEMBRANE PROTEASES IN THE BRAIN. Mass spectrometry (MS) based proteomics offers powerful methods to identify membrane protease substrate candidates in vitro and in vivo. Non-targeted quantification of protein cleavage products in the secretome of brain-derived primary cells or cell lines, as well as cerebrospinal fluid (CSF) are suitable for protease substrate identification. In this context, the secretome comprises all proteins released by cells into body fluids or into the conditioned medium of cultured cells. In all protocols, secreted or membrane proteins are digested with a protease, usually trypsin, to create proteolytic peptides. Articles (Bantscheff et al, 2007; Schulze and Usadel, 2010; Bakalarski and Kirkpatrick, 2016)
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