Sumoylation deficiency due to protein mutation leads to increase in aggregation and apoptosis.
Scientists claim that defects in the ability of a protein known as small ubiquitin-related modifier (SUMO) to keep the protein α-synuclein soluble could play a contributing role in protein aggregation-induced disorders such as Parkinson disease (PD). In vivo and in vitro studies demonstrated a role for the SUMO-based post-translational modification known as sumoylation in protein aggregation and found that sumoylation deficiency increased α-synuclein neurotoxicity.
Jochen H. Weishaupt, Ph.D., at the University of Göttingen and colleagues, report their findings in The Journal of Cell Biology in a paper titled “Sumoylation inhibits α-synuclein aggregation and toxicity.”
Studies have shown that a number of aggregation-prone proteins involved in neurodegeneration are normally sumoylated, while cell-based assays suggest that sumoylation-deficient mutants have an enhanced tendency to aggregate. These findings prompted the University of Göttingen team to more directly measure the aggregation propensity of purified sumoylated and unmodified α-synuclein.
The researchers first confirmed α-synuclein was a target for one of the three SUMO proteins, SUMO2, both in a cell-free system and in a human embryonic kidney cell line HEK293T. They also generated a transgenic mouse model expressing His6-SUMO2 under the control of a neuron-specific promoter and showed in these animals that endogenous α-synuclein efficiently conjugated with SUMO2.
Dr. Weishaupt’s team next looked to characterize the impact of sumoylation on α-synuclein fibril formation under in vitro conditions. Recombinant sumoylated α-synuclein was generated in and purified from bacteria. Its propensity to aggregate was compared with that of unconjugated α-synuclein under the same in vitro conditions.
This showed that while sumoylated α-synuclein didn’t generate fibrils, alpha-synyclein did form fibrils in the presence of free, unconjugated SUMO. Interestingly, the authors report, as long as 50% of the alpha-synyclein was sumoylated, fibril formation was completely blocked, and 10% sumoylation was enough to significantly delay fibril formation. Conversely, the addition of free SUMO in concentrations corresponding to 10, 50, or 100% of the unmodified α-synuclein had no effect on blocking fibrillation.
Using a combination of mutagenesis and mass spectrometry to map two lysine residues on the α-synuclein protein that represented the major SUMO conjugation sites (but had no effect on ubiquitination), allowing them to generate sumoylation-deficient mutants for further study. An indirect fluorescent tagging approach was used to visualize the aggregation propensity and cytotoxicity of the mutant α-synucleins with that of the wild-type (WT) protein.
The results supported the in vitro fibrillation assay, showing that the cells with either of the two mutated, sumoylation resistant α-synucleins were more likely to aggregate and were substantially more toxic. The authors claim this result “further corroborates the hypothesis that the altered aggregation properties and enhanced neurotoxicity indeed represent sumoylation-dependent effects.”
To address the role that SUMO plays in the regulation of α-synuclein-induced toxicity in dopaminergic neurons in vivo, the team then compared the neurotoxicity of human WT α-synuclein with one of the mutant α-synucleins in transgenic rats. Twelve weeks after the animals were injected with an rAAV carrying either the wild-type or mutant human α-synuclein, their brain tissue was evaluated.
Rats injected with the WT α-synuclein vector demonstrated significant loss of dopaminergic vesicular monoamine transporter-type 2 (VMAT2) substantia nigra pars compacta (SNpc) neurons, essentially recapitulating the effects of increased α-synuclein gene dosage and overexpression that occurs in hereditary forms of PD. Critically, though, administering the vector coding for one of the sumoylation-impaired α-synuclein mutants substantially exacerbated the neurodegenerative effect and led to 42% fewer of the relevant neurons surviving.
Interestingly, the researchers note, the two lysine residues representing the major acceptor sites for SUMO on WT α-synuclein lie close to what has previously been identified as the aggregation promoting amino acid stretch. This suggests that normal sumoylation somehow shields the aggregation-promoting region.
“Thus, it is tempting to speculate that modification with SUMO proteins may play a role in the pathophysiology of PD,” they conclude. “To determine whether genetic, environmental, or oxidative stress-induced changes in sumoylation contribute to the disease development will require extensive future investigations.
“In addition to possible—so far, unknown—genetic defects that may alter the equilibrium of reversible sumoylation, the SUMO pathway is known to respond dramatically to many different insults and environmental stresses including heat, oxidative stress, viral and bacterial infection, and ischemia.”