Abstract
Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function.
Highlights
From the ‡Department of Cell Biology and Histology, Academic Medical Centre- University of Amsterdam, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands; §Department of Pathology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305–5324; ¶Netherlands Institute for Neuroscience, Meibergdreef 47, 1105BA Amsterdam, The Netherlands; ʈCenter for Neurogenomics and Cognitive Research, VU University Amsterdam, De Boelelaan 1085, 1081HV Amsterdam, The Netherlands; **Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands; ‡‡Department of Medical Biochemistry and Microbiology, University of Uppsala, Husargatan 3, 75123 Uppsala, Sweden; §§Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333CC Leiden, The Netherlands
The proteomic data revealed that TPP2 inhibition changes the expression of proteins that are linked to the extracellular signal-regulated kinase 2 (ERK2) function, a connection we found to rely on a rapid reduction of phosphorylation level and thereby the activity of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus of neuroblastoma cells
We determined proteomic changes following the application of B6, a newly developed, specific, irreversible TPP2 inhibitor, in parallel with the commercially available TPP2 inhibitor butabindide [23] and compared the data with the proteomic effects induced by stable TPP2 knock down as a reference
Summary
The proteomic data revealed that TPP2 inhibition changes the expression of proteins that are linked to the ERK2 function, a connection we found to rely on a rapid reduction of phosphorylation level and thereby the activity of ERK1 and ERK2 in the nucleus of neuroblastoma cells. To investigate nonspecific and cell adaptive effects of B6, lysates of TPP2-inhibited and control noninhibited cells were incubated with activity-based probes for serine hydrolases and the proteasome, as well as a fluorogenic substrate for leucyl-aminopeptidases to determine the respective peptidase activities.
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