Online Journal Club for April 2009
Our new format includes only relevant articles. If you have articles that you believe should be included in the online journal club, send them to me before the 20th of the month.
This month is a themed journal club, the theme being SUMO, a protein we have started working with. First there will be a breif review of the basics, and then a look at the (scant) literature on this topic in brain ischemia.
SUMO REVIEW (Mostly pilfered from Wikipedia):
Small Ubiquitin-like Modifier or SUMO proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle [1].
SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow for formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.
SUMO family members often have dissimilar names; the SUMO homologue in yeast, for example, is called SMT3 (suppressor of mif two 3). Several pseudogenes have been reported for this gene.
Structure
schematic of human SUMO1 protein made with iMol and based on PDB file 1A5R, an
NMR structure; the backbone of the protein is represented as a ribbon,
highlighting secondary structure; N-terminus in blue, C-terminus in red
The same structure represented with atoms represented
as spheres to show the shape of the protein; human SUMO1, PDB file 1A5R
Function
SUMO modification of proteins has many functions. Among the most frequent and best studied are protein stability, nuclear-cytosolic transport, and transcriptional regulation. Typically, only a small fraction of a given protein is SUMOylated and this modification is rapidly reversed by the action of deSUMOylating enzymes. (Sullythought: could deSUMOylase inhibitors ultimately be neuroprotective?) SUMOylation of target proteins has been shown to cause a number of different outcomes including altered localisation and binding partners. For example, the SUMO modification of hNinein leads to its movement from the centrosome to the nucleus [2]. In many cases SUMO modification of transcriptional regulators correlates with inhibition of transcription [3]. (Sullynote: this could be terribly important.) Refer to the GeneRIFs of the Sumo proteins, e.g. human SUMO1 [4], to find out more.
There are 3 confirmed SUMO isoforms in humans; SUMO-1, SUMO-2 and SUMO-3. SUMO-2/3 show high a high degree of similarity to each other and are distinct from SUMO-1. SUMO-4 shows similarity to -2/3 but it is as yet unclear whether it is a pseudogene or merely restricted in its expression pattern.
SUMO-2/3 modifications seem to be involved specifically in the stress response. SUMO-1 and SUMO-2/3 can form mixed chains, however, because SUMO-1 does not contain the internal SUMO consensus sites found in SUMO-2/3, it terminates these poly-SUMO chains [5]
Structure
SUMO proteins are small; most are around 100 amino acids in length and 12 kDa in mass. The exact length and mass varies between SUMO family members and depends on which organism the protein comes from. Although SUMO has very little homology with Ubiquitin at the amino acid level, it has a nearly identical structural fold.
The structure of human SUMO1 is depicted above. It shows SUMO1 as a globular protein with both ends of the amino acid chain (shown in red and blue) sticking out of the protein's centre. The spherical core consists of an alpha helix and a beta sheet. The diagrams shown are based on an NMR analysis of the protein in solution.
Prediction of SUMO attachment
Most SUMO-modified proteins contain the tetrapeptide consensus motif Ψ-K-x-D/E where Ψ is a hydrophobic residue, K is the lysine conjugated to SUMO, x is any amino acid (aa), D or E is an acidic residue. Substrate specificity appears to be derived directly from Ubc9 and the respective substrate motif. SUMOplot is an online free access software developed to predict the probability for the SUMO consensus sequence (SUMO-CS) to be engaged in SUMO attachment.[6] The SUMOplot score system is based on two criteria: 1) direct amino acid match to the SUMO-CS observed and shown to bind Ubc9, and 2) substitution of the consensus amino acid residues with amino acid residues exhibiting similar hydrophobicity. SUMOplot has been used in the past to predict Ubc9 dependent sites. Seventeen (17) articles have been published so far for the complete list click here.[7] Alternative prediction engines such as SUMOsp are also available [8].
SUMO Conjugation
SUMO conjugation to its target is analogous to that of Ubiquitin (as it is for the other Ubiquitin-like proteins such as NEDD 8). A C-terminal peptide is cleaved from SUMO by a protease (in human these are the SENP proteases or Ulp1 in yeast) using ATP to reveal a di-glycine motif. SUMO then becomes bound to an E1 enzyme (SUMO Activating Enzyme (SAE)) which is a heterodimer. It is then passed to an E2 which is a conjugating enzyme (Ubc9). Finally, one of a small number of E3 ligating proteins attaches it to the protein. In yeast, there are four SUMO E3 proteins, Cst9[9], Mms21, Siz1 and Siz2. While in ubiquitination an E3 is essential to add ubiquitin to its target, evidence suggests that the E2 is sufficient in Sumoylation as long as the consensus sequence is present. It is thought that the E3 ligase promotes the efficiency of sumoylation and in some cases has been shown to direct SUMO conjugation onto non-consensus motifs. E3 enzymes can be largely classed into PIAS proteins, such as Mms21 (a member of the Smc5/6 complex) and Pias-gamma and HECT proteins. Some E3's such as RanBP2 however are neither [10]. Recent evidence has shown that PIAS-gamma is required for the sumoylation of the transcription factor yy1 but it is independent of the zinc-RING finger (identified as the functional domain of the E3 ligases). SUMOylation is reversible and is removed from targets by specific SUMO proteases in an ATP dependent manner. In budding yeast, the Ulp1 SUMO protease is found bound at the nuclear pore, whereas Ulp2 is nucleoplasmic. The distinct subnuclear localisation of deSUMOylating enzymes is conserved in higher eukaryotes[11]
PAPERS: There is almost nothing to date in brain ischemia—and to the
extent there is, Wulf Paschen is all over it.
1. Transient focal cerebral ischemia induces a dramatic activation of small ubiquitin-like modifier conjugation. Wei Yang, Huaxin Sheng, David S Warner and Wulf Paschen. JCBFM (2008) 28, 892–896.
This study was designed to investigate whether small ubiquitin-like modifier (SUMO) conjugation is activated after focal cerebral ischemia. Transient ischemia induced a dramatic increase in SUMO2/3 protein conjugates. The most pronounced changes were found in the parietal cortex. SUMO2/3 conjugation was particularly high in neurons located at the border of the middle cerebral artery territory where sumoylated proteins translocated to the nucleus. Considering the marked effect of SUMO conjugation on the function of target proteins, it is very likely that the postischemic activation of sumoylation has a significant effect on the fate of neurons exposed to transient ischemia.
Link (PDF): http://www.nature.com/jcbfm/journal/v28/n5/pdf/9600601a.pdf
2. Increased protein SUMOylation following focal cerebral ischemia. Cimarosti H, Lindberg C, Bomholt SF, Rønn LC, Henley JM. Neuropharmacology. 2008 Feb;54(2):280-9. Epub 2007 Oct 6.
Stroke is a major cause of death
and disability, which involves excessive glutamate receptor activation leading
to excitotoxic cell death. We recently reported that SUMOylation can regulate
kainate receptor (KAR) function. Here we investigated changes in protein
SUMOylation and levels of KAR and AMPA receptor subunits in two different
animal stroke models: a rat model of focal ischemia with reperfusion and a
mouse model without reperfusion. In rats, transient middle cerebral artery
occlusion (MCAO) resulted in a striatal and cortical infarct. A dramatic increase in SUMOylation by both SUMO-1 and
SUMO-2/3 was observed at 6h and 24h in the striatal infarct area and by
SUMO-2/3 at 24h in the hippocampus, which was not directly
subjected to ischemia. In mice, permanent MCAO resulted in a selective cortical
infarct. No changes in SUMOylation occurred at 6h but
there was increased SUMO-1 conjugation in the cortical infarct and non-ischemic
hippocampus at 24h after MCAO. (SULLYTHOUGHT:
This is critical – I have already started forming a nebulous hypothesis
that hypothermia might accelerate sumoylation in brain
ischemia—see following article.) Interestingly, SUMOylation by SUMO-2/3
occurred only outside the infarct area. In both rat and mouse levels of
3. Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells. Lee YJ, Miyake S, Wakita H, McMullen DC, Azuma Y, Auh S, Hallenbeck JM. J Cereb Blood Flow Metab. 2007 May;27(5):950-62.
Hibernation torpor provides an excellent natural model of tolerance to profound reductions in blood flow to the brain and other organs. Here, we report that during torpor of 13-lined ground squirrels, massive SUMOylation occurs in the brain, liver, and kidney. The level of small ubiquitin-related modifier (SUMO) conjugation coincides with the expression level of Ubc9, the SUMO specific E2-conjugating enzyme. Hypothermia alone also increased SUMO conjugation, but not as markedly as hibernation torpor. Increased SUMO conjugation (induced by Ubc9 overexpression, ischemic preconditioning (PC)+/-hypothermia) was necessary and sufficient for tolerance of SHSY5Y neuroblastoma cells to oxygen/glucose deprivation (OGD) ('in vitro ischemia'); decreased SUMO conjugation (induced by a dominant-negative Ubc9) severely reduced tolerance to OGD in these cells. These data indicate that post-translational modification of proteins by SUMOylation is a prominent feature of hibernation torpor and is critical for cytoprotection by ischemic PC+/-hypothermia in SHSY5Y cells subjected to OGD.