Dataset Information

An Abundance of Free Proteasomal Regulatory (19S) Particles Regulate Neuronal Synapses Independent of the Proteasome

ABSTRACT: The major protein-degradation machine, the proteasome, functions at brain synapses and regulates long-term information storage. Here we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles that assemble to recognize and degrade substrates, were differentially distributed within individual rat cortical neurons. By detecting and quantifying 19 and 20S particles at single-molecule resolution, we discovered a surprising abundance of free regulatory particles (19S) near synapses. Furthermore, we found that the free neuronal 19S binds and deubiquitylates Lys63-ubiquitin, a distinct ubiquitin linkage that does not target substrates to the proteasome. Pull-down assays for Lys63 or Lys48 ubiquitin revealed a significant over-representation of synaptic molecules as Lys63 interactors, while conventional proteasome-related proteins dominated the Lys48 interactome. Inhibition of 19S deubiquitylase (DUB) activity significantly altered spontaneous excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Primary Cell Line Cell, Primary Cell, Cortex, Cell Culture, Brain Cortex Cell Line

SUBMITTER: Julian Langer

LAB HEAD: Julian Langer

PROVIDER: PXD037893 | Pride | 2024-11-26

REPOSITORIES: Pride

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			Action	DRS
	20210315_ChSun_90min_2uL_1-10_2123-R3_05a.raw	Raw
	20210315_ChSun_90min_2uL_1-10_2123-R3_05b.raw	Raw
	20210315_ChSun_90min_2uL_1-10_2123-R3_06a.raw	Raw
	20210315_ChSun_90min_2uL_1-10_2123-R3_06b.raw	Raw
	20210315_ChSun_90min_2uL_1-10_2123-R3_07a.raw	Raw

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An abundance of free regulatory (19S) proteasome particles regulates neuronal synapses.

Sun Chao C Desch Kristina K Nassim-Assir Belquis B Giandomenico Stefano L SL Nemcova Paulina P Langer Julian D JD Schuman Erin M EM

Science (New York, N.Y.) 20230526 6647

The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy, we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles, are differentially distributed within individual rat cortical neurons. We discovered an unexpected abundance of free 19S particles near synapses. The free neuronal 19S particles bind and d ...[more]

PMID: 37228199

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Project description:Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas. It is characterized by the presence of rare Hodgkin and Reed/Sternberg (HRS) cells embedded in an extensive inflammatory infiltrate. Constitutive activation of nuclear factor-kappaB (NF-kappaB) in HRS cells which transcriptionally regulates expression of multiple anti-apoptotic factors and pro-inflammatory cytokines plays a central role in the pathogenesis of cHL (1, 2). In non-stimulated condition, NF-kappaB proteins are rendered inactive by binding to their inhibitors (IkappaB s), which sequester them in the cytoplasm. Stimulation of multiple receptors activates the IkappaB kinase (IKK) complex that phosphorylates IkappaB at two specific serine residues, followed by its ubiquitination and proteasomal degradation, thereby releasing NF-kappaB proteins and allowing their nuclear translocation (3). Recently, two studies provided further insights into the molecular mechanisms of IKK activation upon TNF stimulation (4, 5). Activation of the IKK complex and subsequent NF-kappaB activation requires Lys63 polyubiquitination of RIP1, a kinase which is recruited to the receptor upon TNF stimulation. IKK-ï§ï (NEMO), the regulatory subunit of the IKK complex, specifically recognizes these Lys63-linked polyubiquitins attached to RIP1 and thereby activates IKK and NF-kappaB (4, 5). A20 is an ubiquitin-modifying enzyme that inhibits NF-kappaB activation in succession of tumor necrosis factor (TNF) receptor and Toll-like receptor induced signals (6-8). This enzyme removes Lys63 linked ubiquitin chains from RIP1 and adds Lys48 polyubiquitins to RIP1, thereby targeting this factor for proteasomal degradation, thus explaining the molecular mechanism of NF-kappaB inhibition by A20 (6). A20 likely inhibits NF-kappaB acitivity also by additional means, including interaction with TRAF1 and TRAF2 (9). SNP 6.0 array (Affymetrix) analyses were performed according to the manufacturer's directions on DNA extracted from three Hodgkin cell lines (L1236, HDLM-2, U-HO1), HapMap samples included in the Genotyping Console Software 3.0 were used as references.

Dataset Information

An Abundance of Free Proteasomal Regulatory (19S) Particles Regulate Neuronal Synapses Independent of the Proteasome

Dataset's files

Publications

An abundance of free regulatory (19<i>S</i>) proteasome particles regulates neuronal synapses.

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