UCH-L1 (ubiquitin carboxyl-terminal hydrolase L1) is a protein, which plays an important role in ubiquitin-proteasome system. Many previous reports showed the relation between UCH-L1 and neurodegenerative diseases, diabetes, as well as cancer. However, the mechanism still remains unclear. In the aim to investigate the functions and regulatory mechanism of UCH-L1 in living organism, Drosophila melanogaster model was utilized to examine the role of UCH-L1. This chapter provides a summary on recent findings related to the roles of UCH-L1 based on the model. First, abnormal expression of Drosophila ubiquitin carboxyl-terminal hydrolase (dUCH) leads to the defects on fly tissue development and function. Gain function of dUCH in the eye imaginal discs induced a rough eye phenotype in the adult, partly resulting from induction of caspase-dependent apoptosis, upset of photoreceptor cell distribution and ommatidium apical mispatterning. Interestingly, the dUCH overexpression of induced rough eye phenotype was completely recused by co-expression either Sevenless or Draf of the mitogen-activated protein kinase pathway. Besides, knockdown dUCH in dopaminergic neurons resulted in some Parkinson’s disease—like phenotypes in fly. Taken together, those findings in Drosophila model contributed a significant dUCH in tissue development and function.
- Drosophila melanogaster
- human diseases
- eye development
- anti-dUCH antibody
Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a protein of 223 amino acids (aa), weighs about 24,824 Da, a period lasting for more than 48 half-hour. UCH-L1 is an abundant protein in neurons, accounting for 1–2% of the total protein in the human brain . In addition to the brain, UCH-L1 is also expressed strongly in the peripheral nervous system, including sensory and nervous system activity. UCH-L1 belongs to remove the tagged enzyme (deubiquitinating enzyme (DUB)), an important protein in ubiquitin proteasome system (UPS). UCH-L1 hydrolases the peptide bond between ubiquitins and also plays a function as a ligase when it be in dimer form [2, 3]. UCH-L1 is an enzyme which binds to the polyubiquitin chains and released the single ubiquitin in the ubiquitin proteasome system. However, when UCH-L1 is in binary form, UCH-L1 leads to the formation of a polyubiquitin chain linked through lysine 63 (K63). Although the main activity of UCH-L1 is still unclear, UCH-L1 has been believed to play its role through maintaining a pool of free monomeric ubiquitin which is important for the function of ubiquitin proteasome system . Abnormal function of UCH-L1 leads to the reduction of protein degradation, followed by the accumulation of ubiquitinated protein [5, 6, 7]. UCH-L1, therefore, may relate to many biological processes which dependent to ubiquitination including DNA repair, cell signalling, trafficking, endocytosis and degradation.
In 1998, a missense mutation of UCH-L1 (I93M) was first identified in a German family with Parkinson’s disease (PD) . By contrast, another variant of UCH-L1 (S18Y) was discovered as a factor in the risk reduction of PD . Other studies also found that UCH-L1 was related to abnormal accumulation and aggregation of α-synuclein which leads to formation of Lewy bodies . Furthermore, gracile axonal dystrophy (GAD) mouse which carries a deletion within UCH-L1 gene manifested motor ataxia, axonal degeneration and a reduction in the monoubiquitin level in neurons [10, 11, 12].
On the other hand, many studies indicated that UCH-L1 involved too many types of human cancer . High expression of UCH-L1 was found in many types of cancers such as breast cancer, non-small cell lung cancer [13, 14]. UCH-L1 expression can be self-upregulated via oncogenic β-catenin/TCF activation. The UCH-L1 upregulates oncogenic β-catenin by which feedback regulates the expression of
By contrast, UCH-L1 had been also reported as a tumor suppressor in many other studies. The downregulation of UCH-L1 was observed in various types of cancer such as esophageal cancer, breast cancer, prostate cancer and pancreatic cancer [20, 21, 22, 23, 24]. Reduction in UCH-L1 expression leads to cell proliferation arrest and p53-mediated apoptosis [22, 25].
In humans, the gene coding for UCH-L1 is located in the short arm of chromosome 4 at position 14, from base pair 40,953,685 to 40,965,202, 11,518 base pairs long . In
Drosophila model in the study role of UCH-L1
2.1. Homolog of human ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in
The survey of the
2.2. Generation of anti-dUCH antibody
Drosophila model for studying the UCH-L1 role in tissue development
Being a member of ubiquitin proteasome system (UPS), UCH-L1 is thought to be involved in many different processes in living organisms, such as cell proliferation and differentiation. In
On the other hand, overexpression of dUCH in
Furthermore, dUCH overexpression also caused the upset in distribution of photoreceptor clusters in fly pupal retina ( Figure 5 ).
Interestingly, co-expressing dUCH with Sevenless or Draf in eye imaginal discs could suppress the rough eye phenotype induced by overexpressing dUCH. It is therefore likely that overexpression of dUCH downregulates the MAPK pathway, resulting in impairment of eye development ( Figure 7 ) .
Drosophila model for studying the UCH-L1 role in Parkinson’s disease
UCH-L1 was first linked to PD when mutation UCH-L1I93M was found in two siblings from a family with autosomal dominant PD . Transgenic mice that overexpression of UCH-L1I93M showed an accumulation of α-synuclein with ubiquitin in the brain . UCH-L1-deficient mice showed neuronal loss in the spinal gracile tract and exhibit early development sensory and progressive motor ataxia . However, another mutation UCH-L1S18Y is dedicated that decreased rick in PD by antioxidant and neuron-protective function . Therefore, the mechanism of UCH-L1 still remains unclear. In
2.5. Materials and methods
2.5.1. Fly stocks
Fly stocks were maintained at 25°C on standard food containing 0.7% agar, 5% glucose and 7% dry yeast. Wild-type strain Canton-S was obtained from the Bloomington
2.5.2. Western immunoblot analysis
Wild-type and transgenic adult flies carrying GMR-GAL4 > UAS-dUCH were frozen in liquid nitrogen and homogenized in a solution containing 50 mM Tris-HCl (pH 7.5); 5 mM MgCl2; 150 mM NaCl; 10% glycerol; 0.1% Triton X-100; 0.1% NP-40; 1 mM phenylmethylsulfonyl fluoride; 5 mM β-mercaptoethanol; 10 g/ml each of aprotinin, leupeptin and pepstatin A; and 1 g/ml each of antipain, chymostatin and phosphoramidon. Homogenates were centrifuged, and extracts (200 g of protein) were electrophoretically separated on SDS-polyacrylamide gels containing 10% acrylamide and then transferred to polyvinylidene difluoride membranes (Bio-Rad). The blotted membranes were blocked with TBS/0.05% Tween-20 containing 5% skim milk for 1 h at 25°C, followed by incubation with rabbit polyclonal anti-dUCH at 1:1000 dilution or mouse monoclonal anti-α tubulin (Developmental Studies Hybridoma Bank (DSHB)) at 1:5000 dilution for 16 h at 4°C. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (GE Healthcare Bioscience) at 1:10,000 dilution for 1 h at 25°C. Detection was performed with ECL Western blotting detection reagents (GE Healthcare Bioscience), and images were analyzed with a Lumivision Pro HSII image analyzer (Aisin Seiki).
Larval and adult brains were dissected in cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde at 25°C for 15 min. After washing with 0.3% PBS-T (PBS containing 0.3% Triton-X100) twice, the samples were blocked in blocking solution (0.15% PBS-T containing 10% normal goat serum) at 25°C for 20 min. Samples were then incubated with the following primary antibodies diluted in blocking solution: rabbit anti-
2.5.4. Crawling assay
Male larvae in the early third instar stage were collected randomly and washed with PBS to discard food traces. After that, larvae were transferred to agar plates containing 2% agar with a density of 2–4 larvae per plate. The movement of larvae was recorded by a digital camera for 60 s. The recorded videos were then converted into AVI type by MOV to AVI converter (Pazera Jacek, Poland) and then analyzed by ImageJ (NIH, USA) with wrMTrck plugin (developed by Dr. Jesper Søndergaard Pedersen) to track larval movement and draw motion paths.
2.5.5. Climbing assay
Newly eclosed adult male flies were collected and transferred to conical tubes which have heights of 15 cm and diameters of 2 cm. After that, the tubes were tapped to collect the flies to the bottom, and the length of time to record the movement of flies was 30 s. The procedures were repeated five times and recorded by a digital camera. For all of the climbing experiments, the height which each fly climbed to was scored as follows: 0 (less than 2 cm), 1 (between 2 and 4 cm), 2 (between 4 and 6 cm), 3 (between 6 and 8 cm), 4 (between 8 and 10 cm) and 5 (more than 10 cm). The climbing assay was performed every 5 days until all flies lose their locomotor abilities.
2.5.6. Conclusion and perspective
UCH-L1 was known as a complex and unclear function protein. It has several irrelevant activities as hydrolase and ligase, which are also related to ubiquitin. Previous reports showed that abnormal UCH-L1 functioning, caused by mutations or change in levels of protein expression. Those reports also implied that UCH-L1 could have many negative effects, with impacts on cell proliferation, cell cycling and cell death through activation of many genes [33, 34]. In this chapter, some data compatibly demonstrated that overexpression of dUCH, a homolog of human UCH-L1 in
I am grateful to Professor Yamaguchi Masamitsu and Professor Tran Linh Thuoc for their great supports to our research. I would also like to extend my warm thanks to my students Cao Thi Thuy Trang, Huynh Man Anh and Vuu My Dung for their great contribution on the manuscript.
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