Long viewed as mere trash recyclers inside the cell, proteasomes—at least those in neurons—just got a shiny makeover. According to a paper in the March 13 Nature Structural and Molecular Biology, nearly half of neuronal 20S proteasomes reside not in the cytoplasm, but on the cell surface. From there, these neuronal membrane proteasomes (NMPs) devour intracellular proteins and spit peptides out into the extracellular space. Some of those peptides rapidly induce calcium influx, in part through activation of NMDA receptors on neurons, the researchers claim. Seth Margolis at Johns Hopkins University in Baltimore led the study, with a single graduate student, Kapil Ramachandran, conducting the experiments. The researchers have yet to identify the NMP’s substrates or products; however, if their finding holds up, the discovery of this novel proteasome will likely have wider implications for neuroscience, others said.
Alfred Goldberg of Harvard Medical School, whose work uncovered the function of the proteasome, was impressed. “This work suggests there’s a lot going in the cell membrane that we never dreamed of,” he said. Felix Schweizer of the University of California, Los Angeles, called the paper a tour de force. “Importantly, the authors have uncovered an intriguing and unexpected novel mechanism for communication by neurons. Since communication is crucial for the proper functioning of the brain, any new insight into neuronal communication does potentially bear on disease states. It is unexpected discoveries such as the ones made in this study that are enabled by fundamental research” (see full comment below).
Michael Ehlers of Biogen in Cambridge, Massachusetts, commented that the findings help explain previous observations that proteasome inhibition affects neuronal function with uncanny speed. “Ramachandran and Margolis provide evidence for a surprising and very different role of the proteasome in neurons,” he wrote. “It will be important to understand the molecular complex of the neuronal membrane proteasome in much more detail.”
Plasma Proteasome. Three potential models for how neuronal membrane proteasomes function within or at the plasma membrane. [Image courtesy of Ramachandran and Margolis, Nature Structural and Molecular Biology, 2017.]
The proteasome is the multi-subunit processor that degrades a majority of proteins in the cell. In the largest, 26S, version, ubiquitin-tagged proteins gain entry into the barrel-shaped, 20S, core after opening a regulatory cap (see Coux et al., 1996; Voges et al., 1999). The ubiquitin-proteasome system rids the cell of unwanted proteins, including some amyloidogenic ones implicated in neurodegenerative diseases. It is essential for many neuronal functions, including synaptic remodeling (see Ehlers, 2003).
Blocking the proteasome affects most processes on a timescale of hours to days. However, blocking the proteasome also dampens neurotransmission and alters the strength of the early phase of long-term potentiation within seconds to minutes. These rapid changes are hard to explain based on the complex’s effects on proteostasis (see Dong et al., 2008; Cai et al., 2010; Rinetti et al., 2010).
As a new graduate student in Margolis’s lab, Ramachandran noted a strong reduction in calcium signaling within seconds of blocking the proteasome. To investigate, he took a step back and asked where proteasomes reside in neurons. Using immunogold-electron microscopy, he probed hippocampal brain slices and primary hippocampal neurons for 20S core proteins, and was surprised to find about 40 percent of the 20S proteasomes smack in or near the plasma membrane. Antibodies for the 19S cap did not label the membrane, suggesting no 26S proteasome there. In contrast, HEK293 kidney cells had no membrane-associated 20S proteasomes.
Could these membrane-bound proteasomes somehow explain the rapid cellular responses to proteasome inhibitors? To test this, Ramachandran treated primary mouse cortical neurons with a cell-impermeable proteasome inhibitor called biotin-epoxomicin. This inhibitor is exquisitely specific for the proteasome and only binds to active ones, noted Goldberg. Within 10 to 30 seconds it strongly suppressed the amplitude of calcium transients induced by the GABA antagonist bicuculline. Biotin-epoxomicin also messed with the frequency of the transients, increasing it in half the neurons and decreasing it in a third.
Ramachandran and Margolis reasoned that the NMPs might release peptides into the extracellular space, which then act as neuromodulators. Sure enough, through radioactive protein labeling experiments, Ramachandran found that cultured neurons churned out peptides into the medium and that proteasome inhibitors blocked this exodus. Isolating these peptides, he found they triggered calcium transients when added to primary mouse cortical neurons. Proteinase K abolished this effect. Also, peptides from neurons treated with proteasome inhibitors were unable to trigger calcium influx, suggesting only peptides coming out of the proteasome have this capability. Finally, using various inhibitors of ion channels and neurotransmitter receptors, the researchers determined that NMP peptides triggered the influx of calcium in part via NMDA receptors.
Margolis said this activity of NMP peptides may explain why the proteasome has been implicated in acute neuronal signaling. “Usually we think of signal amplification as happening from outside-in, but this is the opposite,” Margolis said. “Small changes inside the cell translate to massive changes outside.” Margolis added that it is possible the NMPs also work bidirectionally, processing proteins from the outside of the cell inwards, although that has yet to be formally tested.
What holds the 20S proteasome at the membrane? Ramachandran found that antibodies to proteasome subunits readily stained the surface of live neurons, and that the proteasome was sensitive to proteases added to the culture medium. These experiments indicated that at least some of the proteasome protrudes through to the extracellular side of the membrane. Biochemical experiments confirmed that the 20S proteasome clung tightly to membranes in neurons. Moreover, the multipass transmembrane glycoprotein GPM6A/B, expressed primarily in neurons, appears to anchor the NMPs to the membrane. Ramachandran thinks the NMPs, which are hydrophilic, might sit in a pore in the membrane formed by GPM6A/B (see image above).
Why has this membrane proteasome gone unnoticed? Ramachandran said one contributing factor is scientists’ tendency to focus on the capped 26S proteasome rather than uncapped 20S cores. Goldberg added that while all cells contain uncapped 20S cores, no role for these “free agents” had been convincingly demonstrated before. Furthermore, when isolating proteasomes, researchers often throw away membrane fractions from the get-go, Goldberg said. Even with the high resolution of electron microscopy, it is difficult to confirm that a protein is actually associated with the membrane, not merely close by, Ramachandran added.
The findings raise many other questions. How are NMPs regulated? What are their substrates and products? Do NMPs process misfolded proteins implicated in neurodegenerative disease? Finally, do peptides released by NMPs contribute to the heightened neural activity observed during neurodegeneration? How NMPs relate to other degradation pathways in the cell, including the lysosomal and autophagic systems that tend to become overwhelmed during neurodegenerative disease, will be important to understand as well, Ramachandran and Margolis added.
Thomas Behnisch of Fudan University in Shanghai commented that the discovery of the NMP could be important for future and past neurodegenerative disease research. “Reduced proteasome activity has been shown to attenuate some forms of memory,” Behnisch wrote. “However, with the discovery of the transmembrane proteasome, some of the conclusions drawn from previous studies might need to be re-evaluated to take the new findings into account.”
Goldberg noted the proteasome’s essential role in generating antigenic peptides that are then loaded onto major histocompatibility complexes. Much as these peptides allow for constant immunosurveillance of the intracellular environment by lymphocytes, perhaps these NMP-generated peptides provide a continuous stream of information about the activity of synapses or dendrites, he suggested. Whether and how other cells, including neurons and glia, pick up on that information would have to be investigated, he said. Goldberg added that NMP-generated peptides might be externally loaded onto MHC I molecules on neurons or nearby cells. In addition to their classical role in presenting antigens to lymphocytes, MHC I molecules have also been implicated in neuronal development and plasticity and may protect motor neurons from degradation in people who have amyotrophic lateral sclerosis (see Huh et al., 2000; Mar 2016 news). Interestingly, the researchers found that NMPs included subunits specific to the immunoproteasome, which are found in antigen-presenting, hematopoietic cells and specialize in producing antigenic peptides.—Jessica Shugart
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