The Ash Twins Blog

See previous posts on history, symptoms, neurophysiology, and genetics of Angelman Syndrome.

Animal cells have evolved two systems to degrade excess, aged, and abnormal proteins: the ubiquitin-proteasome system and the lysosome (Tai & Schuman 2008). The ubiquitin-proteasome system is responsible for the degradation of most intracellular proteins, while the lysosome degrades most membrane and extracellular proteins.

The ubiquitin-proteasome system mediates essential functions in the nervous system (Tai & Schuman 2008). For example, proteasome inhibitors cause retrograde amnesia in rodents. Ubiquitination and proteasome pathways are essential for the candidate molecular mechanism for memory known as long term potentiation (LTP). Neurons, with their exceptionally large cell volumes and long life (most neurons in the brain are as old as the organism), necessitate especially efficient protein turnover and recycling.

Dysfunction of the ubiquitin-proteasome system has been implicated in the neurodegenerative disorders, such as Alzheimer disease, Parkinson disease, Huntington disease, amyothrophic lateral sclerosis (ALS, a.k.a. Lou Gehrig Disease), and the spinocerebellar ataxias (Lehman et al 2009). In these syndromes aggregates of misfolded proteins collect inside neurons and become resistant to proteasomal degradation. These misfolded proteins may actively inhibit the proteasome, or an upstream mechanism may disrupt proteasome function and permit aggregate formation. Less intuitively, the ubiquitin-proteasome pathway has been incriminated in the autism spectrum disorders (Glessner et al 2009; Lehman et al 2009). This situation makes more sense in light of the recent emergence of the hypothesis that autism is primarily a disorder of protein translation and degradation at dendritic spines (Geschwind 2008, Kelleher & Bear, 2008). Most relevant to the current work, the ubiquitin-proteasome pathway is perturbed in Angelman syndrome.

The proteasome is a 2.5 megadalton macromolecular machine that degrades intracellular proteins (Navon & Clechanover 2009). It consists of a 19S regulatory particle, which binds to ubiquitin and ubiquitin-like moieties to regulate which proteins are degraded, and the 20S core particle, which mediates the amino acid-by-amino acid disassembly of peptides. Proteasomes localize to dendritic spines to precisely regulate synaptic protein content after heightened synaptic activity (Tai & Schuman 2008), and it has recently been shown that this process requires CaM kinase IIα (CaMKIIα) functioning as a scaffolding protein (Bingol et al 2010). As genetic manipulation of CaMKII can rescue the neurological phenotype of the mouse model for AS (van Woerden et al 2007), this molecular connection between CaMKII and the proteasome could represent a essential locus for disease (More in a future post).

Ubiquitination refers to the covalent addition of a 76-amino acid peptide called ubiquitin (called this for historical reasons- was identified in the 1970′s as a protein of unknown function present in all cells) to the ε-amino group of lysine residues on target proteins (Pickart 2001). Key to ubiquitination is the fact that ubiquitin has many lysine residues of its own, so it too can be ubiquitinated, leading to long chains of ubiquitin on targeted proteins. Ubiquitination modulates many key cellular processes by selectively targeting master regulatory proteins for proteasomal degradation. Although degradation is the classic and most common fate for a ubiquitinated protein, ubiquitination can influence proteins in other ways, too, including modulation of ribosomal function, DNA repair, the inflammatory response, and transcription factor binding. Relevant to neuroscientists, ubiquitination can mark a receptor or ion channel for endocytosis (Hicke 1999). Whether ubiquitination signals for degradation or something else probably depends on the length of the polyubiquitin chain, the subcellular localization of the substrate, and the topology of the moiety formed by the ubiquitin-substrate complex (Pickart 2001). For example, polyubiquitin chains linked by K48-G76 bonds are usually destined for proteasomal degradation, whereas chains linked by K63-G76 bonds are implicated in intracellular signaling. Deubiquitinating enzymes (DUBs) depolymerize ubiquitin chains, and can thus reverse a protein’s degradation-fate if there are no proteasomes in close proximity and/or the ubiquitin chain is not long enough (Tai & Schuman 2009).

A dozen ubiquitin-related covalent protein modifiers have been identified in addition to ubiquitin (Jentsch & Pyrowolakis 2000). Most have high homology to ubiquitin but instead of mediating degradation are involved in a wealth of other cellular processes. Sumoylation with SUMO-1 (small ubiquitin-related modifier), for example, can direct proteins to translocate into the nucleus at specific points during the cell cycle. Rubylation with RUB (related to ubiquitin) stabilizes scaffolding proteins for the formation of ubiquitination complexes.

Ubiquitination occurs in a three-step process catalyzed by three ligases: E1, E2, and E3 (Pickart 2001). First, E1 ligase forms a thiol ester with ubiquitin’s G76 carboxyl group, activating the ubiquitin C-terminus. Next, E2 ligase transfers the activated ubiquitin from E1 to E3. Last, E3 ligase catalyzes the covalent linkage of ubiquitin to a protein substrate. Polyubiquitin chain formation sometimes requires an additional E4 ligase. The organization of ligases is hierarchical: There is one E1; several E2s, each of which can load several E3s; and hundreds of E3s, each of which target a few substrate proteins.

E3 ligases therefore determine which proteins are ubiquitinated by recognizing highly specific structural motifs on their target proteins.  The ubiquitination signal recognized by a given E3 is frequently a short primary amino acid sequence. Unfortunately, high-throughput methods for identifying E3 substrates are unavailable, and the specific targets for most E3s are undefined (Tai & Schuman 2008).

E3 ligases subdivide into two classes based on their catalytic domain, the HECT ligases and RING ligases. The HECT (Homologous to E6-AP Carboxy Terminus) domain is a highly conserved E2-binding site. RING ( Really Interesting New Gene) refers to a double-zinc cross-brace structure that also specifically binds E2. The domains, although structurally quite distinct, do not appear to differ significantly in function (Pickart 2001).

By incredible coincidence, ubiquitin E3A ligase (UbE3A), the protein implicated in Angelman Syndrome, was the first HECT ligase to be studied in detail. It was discovered as the E3 ligase which is co-opted by human papillomavirus (HPV) to ubiquitinate and degrade the antineoplastic protein p53. The HPV protein which binds UbE3A to degrade p53 is called E6, and thus UbE3A is also called E6-associated protein (E6-AP).

Until very recently, the normal physiological protein substrates of UbE3A (i.e. when it is not in a complex with E6) were for the most part unknown. The Howley lab showed that HHR23A and the Src kinase Blk are ubiquitinated by UbE3A (Kumar et al 1999; Oda et al 1999), and Banks and Kuhne demonstrates that Mcm7 is ubiquitinated by UbE3A as well (1998).  In the March 2010 issue of Cell, Greer et al. identify the ubiquitination signal of UbE3A and report its protein substrates. In the key experiment, a hemagglutinin-tagged ubiquitin knock-in mouse was crossed with the UbE3A knockout and WT mice. Brain lysate was incubated with anti-hemagglutinin antibody to pull down ubiquitin-bound proteins, which were then identified by mass spectrometry. Any proteins that were pulled down in the WT prep but not the UbE3A prep were likely ubiquitinated specifically by UbE3A. Three novel protein substrates were identified: Arc, Ephexin5, and Sacsin.

UbE3A recognizes these proteins and attaches a ubiquitin to them, which causes them to be degraded. Loss of UbE3A, as in Angelman Syndrome, would prevent these proteins from being degraded and lead to an excess of them in cells. In the next post, I describe the function of these proteins in greater detail to help us think about what the effects of their faulty degradation would be.

Bingol B, Wang CF, Arnott D, Cheng D, Peng J, Sheng M. (2010) “Autophosphorylated CaMKIIalpha acts as a scaffold to recruit proteasomes to dendritic spines.” Cell. 140(4):567-78.

Geschwind DH (2008)“Autism: many genes, common pathways?” Cell. 135(3):391-5.

Glessner JT, Wang K, Cai G (2009) “Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.” Nature 459:569–573

Greer PL, Hanayama R, Bloodgood BL, Mardinly AR, Lipton DM, Flavell SW, Kim TK, Griffith EC, Waldon Z, Maehr R, Ploegh HL, Chowdhury S, Worley PF, Steen J, Greenberg ME. (2010) “The Angelman Syndrome protein Ube3A regulates synapse development by ubiquitinating arc.” Cell. 5;140(5):704-16.

Hicke L. (1999) “Gettin’ down with ubiquitin: turning off cell-surface receptors, transporters and channels.” Trends Cell Biol.9(3):107-12. Review.

Jentsch S & Pyrowolakis G (2000) “Ubiquitin and its kin: how close are the family ties?” Trends Cell Bio 10.

Kelleher RJ, Bear MF. (2008) “The autistic neuron: troubled translation?”  Cell.  135(3):401-6.

Kuhne C, Banks L. (1998) “E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box.” J. Biol. Chem. 273:34203–9

Kumar S, Talis A, Howley PM. (1999) “Identification of HHR23A as a Substrate for E6-associated Protein-mediated

Ubiquitination” J. Biol. Chem. 274:18785–92

Lehman NL (2009) “The ubiquitin proteasome system in neuropathology.” Acta Neuropathol. 118(3):329-47.

Navon A Ciechanover A (2009) “The 26 S Proteasome: From Basic Mechanisms to Drug Targeting” J. Biol. Chem. 2009 284: 33713-33718.

Oda H, Kumar S, Howley PM. (1999). “Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination” PNAS 96:9557–62

Pickart CM (2001) “Mechanisms underlying ubiquitination.” Annu Rev Biochem. 70:503-33. Review.

Tai & Schuman (2008) “Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction” Nat Rev Neuro.

van Woerden, G.M., Harris, K.D., Hojjati, M.R., Gustin, R.M., Qiu, S., de Avila Freire, R., Jiang, Y.H., Elgersma, Y. and Weeber, E.J. (2007) “Rescue of neurological deficits in a mouse model for Angelman syndrome by reduction of alphaCaMKII inhibitory phosphorylation.” Nat. Neurosci., 10, 280–282.