טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBajorek Monika
SubjectThe Proteasome as a Rate Limiting Component of the
Ubiquitin-Proteasome Pathway
DepartmentDepartment of Biology
Supervisor Professor Michael Glickman


Abstract

Proteolysis of most eukaryotic proteins takes place within the 20S core particle of the proteasome, a four-ringed barrel-shaped enzyme. Free proteasome 20S CP are found in an auto-inhibited state in which the N-termini of neighboring alpha subunits block access to the channel.

In order to map the relative contributions of different residues to channel gating, we constructed a set of mutations in each of the participating a tails. We conclude that one hydrogen bond between a3 and a4 and van der Waals interactions among the three a subunit tails, a3, a4, a2 define the closed stabilized conformation. Other a subunit tails seem to play a role in 20S and 19S interaction. Our work points to a2 tail as the main player in 20S-19S interaction, therefore critical for stabilizing the open 20S conformation. We show clearly that the N terminal a subunits tails  play a critical role in stabilizing the open and closed 20S conformation, and it is precisely their difference that are integral to their function.

Intracellular degradation is usually thought to be regulated predominantly at the level of the E3s that select substrates for degradation. However, we have shown that proteolysis at the level of the proteasome is a regulated step that can be enhanced or inhibited under certain conditions. By deleting the N terminal tails of different 20S a subunits, we could generate channel mutants which exhibit a panel of effects, from faster (a3/a7DN) to slower (a2DN) protease rates. Moreover, we show that channel gating is responsible for adaptable proteasome activity. Channel gating appears to be a regulated process that can be enhanced or inhibited under certain conditions.

We have also shown that under severe stress conditions a dramatic decrease in proteasome-dependant proteaolysis occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle and 19S regulatory particle components.

Using our faster proteasome mutant a3/a7DN, we have also identified that gating serves an important role in maintaining proteolysis quality control: influencing the rate at which substrates enter and products exit the CP, altering the specificity towards selective peptide bonds thus changing the nature of peptides produced.  The peptides generated by the mutant differ in both C and N terminus and the diversity of peptides in lengths suitable for peptides presentation is greater. Finally we propose that alpha subunits play a role in quality control of proteolysis by the beta subunits.