An expansion of homopolymeric amino acid (HPAA) tract is found in a group of at least ten inherited neurodegenerative diseases. The most common is Spinocerebellar ataxia type 3 (SCA3) also known as Machado-Joseph disease. SCA3 is the predominant late adult onset disease that presents with symptoms including tremor, impaired balance and premature death. The disease protein ataxin-3 (Atx3) is mutated and its normal repeat length of 14-37 polyglutamine residues increases to a number of 54-84 glutamine residues. The characteristic feature of SCA3 is the formation of intranuclear aggregates causing neuronal cell death and degeneration where only a specific subset of neurons is affected. The C-terminal fragments of Atx3 protein found in the patient's brain lysates were shown to be more toxic and with greater tendency for aggregation, which was confirmed in cellular and animal models. Proteases involved in the processing and propagation of Atx3 toxic fragment remain to be identified. Nina Vidergar’s work aimed to understand the mechanism of degradation of ataxin-3 (Atx3) and its mutants with cysteine cathepsins and the potential role of the C-terminal truncated forms of ataxin-3 with higher tendency to aggregate formation and trigger of cell death. Using standard molecular cloning protocols we generated different Atx3 mutants and its C-terminal fragments with various poly-Q repeat lengths. Cellular models of neuroblastoma SH-SY5Y cells expressing mutated Atx3 and its fragments were more prone to aggregation comparing to the wild type. The constructs of Atx3, its mutant and C-terminal fragments were expressed and isolated from host bacteria E. coli (DE3)pLysS for biochemical in vitro studies and further cleavage experiments. The experiments of Atx3 cleavages with cathepsins B, L and K showed that cathepsins process Atx3. With N-terminal-sequencing we have identified cathepsin L as likely protease responsible for the generation of the toxic fragment. The colocalization of Atx3 and cathepsin L was confirmed by immunofluorescent microscopy. SCA3 is a late-onset disease and age-related phenomena such as caspase-, cysteine protease- and calpain activation including the decreased chaperone activity could contribute to the disease initiation. Therefore, the identification of cleavage sites in recombinant human Atx3 may contribute to the understanding of mechanism of proteolysis and its potential link to pathophysiology.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 25904423Traditional international scientific meeting in the field of proteolysis under normal and pathological conditions (Portorož, Slovenia, September 22-26, 2012).
B.01 Organiser of a scientific meeting
COBISS.SI-ID: 263241984In this lecture, the project leader, V. Stoka, reported for the first time, a new concept of neurodegeneration as functional and structural protein networks.
B.05 Guest lecturer at an institute/university
COBISS.SI-ID: 23599911In this lecture, the project leader, V. Stoka, who firstly proposed the lysosomal pathway of apoptosis in 2001, presented a more comprehensive view of the pathway described as a "topological network".
B.04 Guest lecture
COBISS.SI-ID: 23599655Protein-protein interactions define the molecular networks within an organism, whereas a disruption on the later may lead to disease. In this work, we presented a structural and functional network to exemplified this concept on the kallikrein-kinin (KKS) and renin-angiotensin (RAS) sistems. They represent two highly regulated proteolytic systems which participate in several physiological and pathological processes i.e. neurodegeneration, cardiovascular and renal homeostasis, growth and development and inflammation.
B.04 Guest lecture
COBISS.SI-ID: 24648743