טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentInbar Duek
SubjectA Shifting Ubiquitin Landscape at Mitochondria: The Effect
of Protein Degradation Pathways
DepartmentDepartment of Biology
Supervisor Full Professor Glickman Michael
Full Thesis textFull thesis text - English Version


Abstract

Proteins  are  the  building  blocks  of  the  cell  and  therefore they  are  under  tight regulation throughout their synthesis and function. The two main processes for protein

degradation  in  the  cell  are  proteasomal  degradation  and  lysosomal  degradation.

Ubiquitin  is  a  76  amino  acid  protein  that  serves  as  a  signal molecule  for  both pathways. Abnormal or misfolded proteins, as well as proteins that have fulfilled their

purpose,  are  being  ubiquitinated  and  degraded  by  the  ubiquitin  proteasome  system (UPS).  Autophagy is one of the lysosomal pathways, by which bulk cytoplasm is engulfed by an isolated membrane. Originally, autophagy was discovered as a non-specific starvation coping mechanism. Nowadays it is clear that this mechanism can also degrade cellular components in a specific way. Ubiquitin signals proteins to localize near the elongating membrane of the autophagosomes, which enables their degradation by the lysosome. The autophagy process can lead to degradation not only of proteins but of whole organelles as well. Mitophagy is a pathway by which mitochondria are being degraded by the lysosome. The mitochondria are a unique organelle in the cell. Mitochondria takes part in many

biological  pathways,  but  its  main  role  is  to  produce  energy through oxidative phosphorylation. One of the by-products of oxidative phosphorylation are ROS (reactive oxygen species). ROS can oxidize proteins and cellular component thus damaging their structure and function.  Being the main source for ROS formation, mitochondria are highly sensitive to oxidative stress and ROS. Mitochondria dysfunction was found in early stages of many neurodegenerative diseases such as Alzheimer disease, Parkinson disease and Huntington disease. In addition, recent studies claim involvement of ubiquitin and the UPS in aging and neurodegeneration as well. Several studies on these subjects have identified elevation in ubiquitination or proteasomal dysfunction.  Moreover,  in  the  last  two  decades  it  was  established  that mitochondrial proteins are being ubiquitinated for regulatory purposes.  In this study, we   investigated the change   in   the ubiquitination pattern of the mitochondria, upon inhibition of the major proteolytic pathways. We  were  able  to  extract  highly  enriched  mitochondria  from cells  treated  with autophagy  inhibitor Bafilomycin  A1 or proteasome  inhibitor MG132. We discovered that  autophagy  inhibition  with  Bafilomycin  A1  did  not  cause any  changes  in  the ubiquitin   levels   at   the   mitochondria.   Moreover,   it   had   a mild   effect   on   the mitochondria   morphology.   On   the   other   hand,   proteasome   inhibition   causes mitochondria fragmentation and an increase of almost 25 fold in the ubiquitin levels at the mitochondria.  We  were  able  to  detect  an  increase in  numerus  linkage  types: K11,  K27,  K29,  and  K48.  Based  on  our  results  we  were  able  to  build  a  model  to describe the mitochondria reaction to the proteasome inhibitor MG132. According to our  model,  the  mitochondrial  sphere  is  expanded  due  to  accumulation  of  ubiquitin, UPS components including proteasome, and regulatory proteins such as chaperons in the   mitochondrial   fraction.   Lastly,   we   identified   induction   of   autophagy   upon proteasome inhibition by MG132.  This  suggests  a link  between  the  two major proteolytic  pathways  in  the  cell,  where  autophagy  is  induced  in  order  to  remove excess or damaged mitochondria.