Cerqueira 2016 Abstract Mito Xmas Meeting Innsbruck

The Deubiquitinating (DUB) protein Ubiquitin carboxyl- terminal hydrolase L1 (UCH-L1) regulates Mitofusin-2 levels and  mitochondrial function in mammalian cell lines and in D. Melanogaster.

Link:

Cerqueira FM, von Stockum S, Lunsbury P, Ziviani E, Shirihai O (2016)

Event: Mito Xmas Meeting 2016 Innsbruck AT

The ubiquitin carboxyl- terminal hydrolase L1 (UCH-L1) is a cytosolic deubiquitinating (DUB) protein, highly expressed in neurons and beta-cells, which substrates are not well known. UCH-L1 is linked to a familiar case of Parkinson`s Disease (PD), therefore its gene is classified as PARK5.  Curiously, UCH-L1 is being associated to type 2 Diabetes, and it is found reduced in beta cells from Diabetic donors. Although mitochondrial dysfunction is central in PD and Diabetes, mitochondrial proteins were not reported, so far, to be a target of UCH-L1. Given the similarities and conserved pathways between beta cells and neurons, we proposed to investigate the role of UCH-L1 on mitochondrial dynamics and function in neuroblastoma SY5Y-SH and beta cell line INS1 and we are using D. Melanogaster as an in vivo model. Knocking down UCH-L1 in both cell lines SY5Y-SH and INS1, resulted in mitochondrial fragmentation, reduced mitofusin-2 levels, and higher mitochondrial spare capacity, without any increment in mitochondrial mass.  Interestingly, the knockdown of UCH, the UCH-L1 homologue, in  D. Melanogaster reduced Marf levels, and resulted in significantly higher maximum respiratory capacity in isolated mitochondria, suggesting changes in respiration are not coupled to any direct morphology alteration promoted by this DUB.

To test if UCH-L1 activity affects Mfn2 ubiquitiation, we quantified Mfn2 ubiquitination levels in cells where UCH-L1 was depleted. Upon UCH-L1 KD, Mfn-2 levels were drastically reduced. The addition of the proteasome inhibitor MG-132 prevented Mfn2 degradation in UCH-L1 KD cells. Under these conditions we measured increased levels of ubiquitylated Mfn2 compared to the control cells (non-UCH-L1 depleted, also treated with MG-132). Pull down assays showed an interaction between Mfn-2 and UCH-L1 when cells were treated with MG-132 or when cross-linking with formaldehyde was performed, suggesting the interaction between both molecules is transient and enhanced under circumstances of accumulation of ubiquitylated Mfn2 (i.e. MG-132 treatment).

This study suggests UCH-L1 is a DUB which regulates mitochondrial function and Mfn2 is a potential target.

Labels: MiParea: Genetic knockout;overexpression  Pathology: Parkinson's 

Organism: Hexapods 

Preparation: Isolated mitochondria 

Event: Poster 

Affiliations

Cerqueira FM(1), von Stockum S(2), Lunsbury P(3), Ziviani E(2), Shirihai O(1)

1. National Inst Biotechnology Negev & Ben-Gurion Univ, Israel; Univ California, CA, USA
2. Padova Univ, Italy 
3. Harvard Univ, MA, USA