טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentWeinstein Neta
SubjectCharacterization of ARTS Protein and the Effects Of Pro-
Apoptotic Stimulations on its Expression in
Cortical Neurons
DepartmentDepartment of Medicine
Supervisor Professor Emeritus John Finberg
Full Thesis textFull thesis text - English Version


Abstract

ARTS is a septin-like protein derived from alternative splicing of the H5/PNUTL2/hCDCrel2a/2b gene, and has a unique 27 amino acid sequence in its C-terminus which does not exist in other septins. Human ARTS is located in the mitochondria and upon apoptotic stimuli is released to the cytosol, binds XIAP and causes caspase activation and apoptosis. This study was directed at obtaining a better understanding of ARTS function in the brain, since it is highly expressed in neurons but its role there is unknown. Rat primary cortical neuronal cultures were used.

I first showed that the polyclonal anti-ARTS antibody is co-reactive to rat ARTS, even though it was produced using as antigen a fragment of the C-terminus of the human peptide. Human ARTS is a 32 kDa protein but appears as a 28 kDa form in the brain. Peripheral rat ARTS (cardiomyocytes) has a 28 kDa form in addition to the 22 kDa form seen in the brain. The reason for this difference is unknown. Proteasome and protease inhibition did not result in a larger molecular weight form. Sequencing of ARTS cDNA and subsequent prediction of amino acid composition showed 88 % identity between rat and human ARTS. The first methionine location in rat ARTS was different from human ARTS resulting in a smaller predicted protein size of ~20 kDa.

Proteasome inhibition caused elevation in ARTS levels, which confirms previous findings that ARTS is degraded through ubiquitination.

ARTS involvement in neuronal apoptosis was analyzed by treating primary neuronal cultures with various apoptotic inducers - etoposide, ara-C, glutamate and B27 deprivation. All treatments caused apoptosis, but most of them did not affect ARTS in a significant manner. Glutamate and ara-C were the only treatments which resulted in a significant increase in ARTS expression, so that under strong apoptotic conditions ARTS might be involved in the apoptotic pathway. ARTS may be bound to the mitochondria in a non-active form, and released upon apoptotic stimulation.

An immuno-fluorescence analysis using neu-N co-localization showed that all neurons express ARTS. Analysis with BrdU showed that ARTS is not expressed in "new-born" neurons, but only in differentiated neurons. Immuno-fluorescence in rat neurons showed that nARTS is localized both to the nucleus and the cytoplasmatic compartment.

ARTS may have a role in the brain that does not involve apoptosis, since neurons express high levels of ARTS but usually do not show a high level of apoptosis.