Ubiquitin-mediated proteolysis

Presented by:- ARPIT JOSHIBIOCHEMISTRY

Cell biology & Genetics

Department of bioscienceS.P university.

UBIQUITIN-MEDIATED

PROTEOLYSIS

ubiquitin PDB 1TBE

Content :-

• What is ubiquitin mediated proteolysis?• Who discovered?• Pathway• Site of intracellular degradation• Functions• Involve in the regulation of the cell cycle

Ubiquitin• Ubiquitin was first isolated

from bovine thymus but later found in many different tissues and in many organisms.

• Ubiquitin is a highly conserved protein

• Ubiquitin is composed of 76 amino acid

• Attachment site to target protein on ubiquitin is C-terminus

• Bond is formed to side chain of Lys of target protein

• Attachment is performed by array of enzymes (E1, E2, E3, E4).

Proteolysis• Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. 

• In general, this occurs by the hydrolysis of the peptide bond, and is most commonly achieved by  cellular enzymes called proteases,  but may also occur by intramolecular digestion, as well as  by  non-enzymatic  methods  such  as  the action of mineral acids and heat.

Scientist who discovered ubiquitin mediated proteolysis

Avram Hershko Aaron Ciechanover Irwin Rose

Nobel prize in chemistry, 2004

Ubiquitin mediated proteolysis

• A  process  where  an  enzyme  system  tags  unwanted 

proteins  with  many  molecules  of  the  76-amino  acid 

residue protein ubiquitin.

• The  tagged  proteins  are  then  transported  to  the 

proteasome(a  large multisubunit  protease  complex)       

where they are degraded into small peptides.

 

THE UBIQUITIN-PROTEASOME PATHWAY

Unwanted protein

Enzymes of the pathway• E1:

– ubiquitin-activating enzyme.– Activates the carboxy terminus of

ubiquitin by reaction with ATP.– exists as two isoforms of 110- and 117-kDa, are

found in both the nucleus and cytosol.– In mammals there is a single E1.

• E2:– Ubiquitin-conjugating enzymes.– E2s are a super family of related proteins.

There are eleven E2s in yeast, and 20-30 E2s in mammals.

• E3s:– Ubiquitin-protein ligases.– E3s play a key role in the ubiquitin pathway,

as they are responsible for the selective recognition of protein substrates.

• E4:– catalyzes the efficient polymerization of

very long polyubiquitin chains, it has been characterized in yeast.

THE UBIQUITIN-PROTEASOME PATHWAY

Unwanted protein

Three enzymes are involved, designated E1, E2 & E3.

Initially the terminal carboxyl group of ubiquitin is joined in a thioester bond to a cysteine residue on Ubiquitin-Activating Enzyme (E1). This is the     ATP-dependent step.

The ubiquitin is then transferred to a sulfhydryl group on a Ubiquitin-Conjugating Enzyme (E2).

A Ubiquitin-Protein Ligase (E3) then promotes transfer of ubiquitin from E2 to the e-amino group of a Lys residue of a protein recognized by that E3, forming an isopeptide bond. There are many distinct Ubiquitin Ligases with differing substrate specificity. • One E3 is responsible for the N-end rule. • Some are specific for particular proteins. 

ubiquitin C S

O

Cys E2 H2N Lys protein to be degraded

ubiquitin C

O

HS Cys E2N Lys protein to be degraded

H

+

E3

+

(Ubiquitin-Protein Ligase)

H2N COO

destruction box

chain of ubiquitins

Primary structure of a protein targeted for degradation

More ubiquitins are added to form a chain of ubiquitins. The terminal carboxyl of each ubiquitin is linked to the   e-amino group of a lysine residue (Lys29 or Lys48) of the adjacent ubiquitin. A chain of 4 or more ubiquitins targets proteins for degradation in proteasomes. 

H2N COO

destruction box

chain of ubiquitins

Primary structure of a protein targeted for degradation

Some proteins (e.g., mitotic cyclins involved in cell cycle regulation) have a destruction box sequence recognized by a domain of the corresponding Ubiquitin Ligase.  

Ubiquitin Ligases (E3) mostly consist of two families:

1) HECT (Homologous to the E6-AP Carboxyl Terminus)-domain containing a conserved Cys• Some Ubiquitin Ligases have a HECT domain containing a conserved Cys residue that participates in transfer of activated ubiquitin from E2 to a target protein. 

2) RING finger-domain Cys & His residues are ligands to two Zn++ ionsstabilizes a molecular scaffold

Some Ubiquitin Ligases contain a RING finger domain in which Cys & His residues are ligands to 2 Zn++ ions.A RING (Really Interesting New Gene) finger is not inherently catalytic. It stabilizes a characteristic globular domain conformation that serves as a molecular scaffold for residues that interact with E2.

Combinatorial nature of ubiquitination

• Ubiquitin—mediated degradation of cytosolic and membrane proteins occurs in the cytosol and on the cytosolic face of the ER membranes.

Site of intracellular degradation

The structure of proteasome

The 20S proteasome core complex encloses a cavity with  3 compartments joined by narrow passageways. 

Protease activities are associated with 3 of the b subunits, each having different substrate specificity. 

20 S Proteasome (yeast) closed state

two views PDB 1JD2

bb

1. One catalytic b-subunit has a chymotrypsin-like activity with preference for tyrosine or phenylalanine  at the P1 (peptide carbonyl) position.(hydrophobic a.a)

2. One has a trypsin-like activity with preference for arginine or lysine at the P1 position.(positive charged a. a)

3. One has a peptidoglutamyl peptidase like activity with preference for glutamate or other acidic residue at the P1 position. (negative charged a.a)

Different variants of the 3 catalytic subunits, with different substrate specificity.

Degradation in the proteomes

The poly-ubiquitinated protein is recognized by the 26S proteasome, unfolded and degraded. The ubiquitin molecules are recycled.

•Length of produced peptides: 3-23 amino acids

•Average length of peptides: 7-9 amino acids

•Peptide composition of given protein stays constant

•Protein is completely degraded before import of next protein

•Peptides produced by proteasome are further degraded by other proteases and amino peptidases.

• Proteasome and immune system function:• Peptides of 8-9 amino acids in length are

transported to the cell surface via the ER presented on the cell surface via MHC class I – molecules

Processing via the proteasome

DEUBIQUITINATION

De-ubiquitinating

Deubiquitination enzymes

• Eukaryotic cells also contain DUBs (Deubiquitinating enzymes), which are encoded by the UCH (Ubiquitin Carboxyl-terminal Hydrolases) and the UBP (UBiquitin-specific Processing proteases) gene families.

• UCHs are relatively small proteins (< 40-kDa);in contrast, UBPs are 50-250-kDa proteins and constitute a large family.

• Genome sequencing projects have identified more than 90 DUBs .

Ubiquitin mediated proteolysis and its many biological functions

At the end of summer of 1979 in Fox Chase Cancer Center, Philadelphia. Seated left to right: Avram Hershko, Sandy Goldman, Jessie Warms, Hanna Heller,Standing left to right: Zelda Rose, Arthur Haas, Aaron Ciechanover, Mary Williamson, Irwin Rose, Keith Wilkinson and Leonard Cohen (last three people standing on theright side not identified)

Reference• Advanced information on the Nobel Prize in

Chemistry, 6 October 2004,kungl.vetenskapsahedemien,the royal swedish academy of science Information Department, P.O. Box 50005, SE-104 05 Stockholm, Sweden.

• Biochemistry- Lubert Stryer• HARPER’S ILLUSTRATED BIOCHEMISTRY (28TH

EDITION) by Robert murray,david A.bender,peter j kennekky,victor w rodwell,p.antony weil.