This reveals a key functional importance of Bsn4 interaction with the C-terminus of PSMB4

This reveals a key functional importance of Bsn4 interaction with the C-terminus of PSMB4. increased proteasomal Nitisinone activity in the synaptic fractions prepared from brains of bassoon knock-out mice. Finally, increased activity of proteasome and lower expression levels of synaptic substrates of proteasome could be largely normalized upon expression of PSMB4-interacting fragments of bassoon in neurons derived from bassoon deficient mice. Collectively, we propose that bassoon interacts directly with proteasome to control its activity at presynapse and thereby it contributes to a compartment-specific regulation of neuronal protein homeostasis. These findings provide a mechanistic explanation for the recently described link of bassoon to human diseases associated with pathological protein aggregation. Graphic Abstract Presynaptic cytomatrix protein bassoon (Bsn) interacts with PSMB4, the 7 subunit of 20S core proteasome, via three independent interaction interfaces. Bsn inhibits proteasomal proteolytic activity and degradation of different classes of proteasomal substrates presumably due to steric interference with the assembly of 20S core of proteasome. Upon Bsn deletion in neurons, presynaptic substrates of the proteasome are depleted, which can be reversed upon expression of PSMB4-interacting interfaces of Bsn. Taken together, bsn controls the degree of proteasome degradation within the presynaptic compartment and thus, contributes to the regulation of synaptic Nitisinone proteome Electronic supplementary material The online version of this article (10.1007/s00018-020-03590-z) contains supplementary material, which is available to authorized users. test Expression of PSMB4-interacting fragments of Bsn normalizes increased proteasome activity in BsnGT Bsn-deficient mice display systemic phenotypes including epilepsy and might be also prone to neurodegeneration [2, 13, 41, 49]. To determine whether increased proteasome activity is a direct result of bassoon deficiency or a secondary effect of systemic phenotypes described in the animals deficient for Bsn expression, we tested chymotrypsin-, trypsin and caspase-like peptidase activities using fluorogenic specific peptidase substrates in cultured cortical neurons derived from newborn BsnGT animals and their WT littermates. All tested peptidase activities associated with proteasome were significantly increased in cell lysates from cultured neurons from BsnGT animals compared with their WT littermates (Fig.?7A and B-D carmine box; chymotrypsin-like activity: BsnGT-EGFP: 120??3, trypsin-like activity: BsnGT-EGFP: 121??5 and caspase-like activity: BsnGT-EGFP: 115??2% of WT-EGFP). Similarly to HEK293T cells (Fig.?4aCc), expression of fragments Bsn2 and Bsn4 significantly decreased all tested proteasome-associated peptidase activities in WT neurons indicating a physiological role of the PSMB4-interacting interfaces of Bsn in the Nitisinone regulation of the endogenous Nitisinone neuronal proteasome (Fig.?7bCd, white and gray boxes, chymotrypsin-like activity: WT-Bsn2: 72??3, WT-Bsn4: 74??2 of WT-EGFP, WT-EGFP epo: 15??4; trypsin-like activity: WT-Bsn2: 69??2, WT-Bsn4: 70??1, WT-EGFP epo: 24??2; caspase-like activity: WT-Bsn2: 74??4, WT-Bsn4: 73??4% of WT-EGFP, WT-EGFP epo: 17??2, all in ?% of WT-EGFP). Strikingly, expression of Bsn2 and Bsn4 effectively reduced and fully normalized the elevated peptidase activities in BsnGT neurons (Fig.?7bCd, pink and red boxes chymotrypsin-like activity: BsnGT-Bsn2: 79??2, BsnGT-Bsn4: 81??1; trypsin-like activity: BsnGT-Bsn2: 84??4, BsnGT-Bsn4: 82??5; caspase-like activity: BsnGT-Bsn2: 79??3, BsnGT-Bsn4: 75??1, all Nitisinone in ?% of WT-EGFP). This data confirms a key role of Bsn-PSMB4 multivalent interaction in the regulation of proteasome activity in neurons. Moreover, it strongly indicates that increased proteasome activity detected upon deletion of Bsn can be attributed to this molecular interaction. Open in a separate window Fig.?7 Overexpresion of the PSBM4-interacting interfaces of Bsn in BsnGT cultures rescues the proteasome activity levels. a Chymotrypsin-, trypsin- IKBKB and caspase-like activities are elevated in BsnGT cortical neurons as compared to the neurons from their WT littermates. Chymotrypsin- (b), trypsin- (c) and caspase-like (d) activities were significantly reduced upon expression of PSMB4-interacting.