26S Proteasome and Ubiquitin

Aliyah Jackson, Kathy Duong, Allison Weber,

Kaitlyn Greenwood, Van Hopkins
BIOL 324
March 9, 2020
 The 26S Proteasome is a protein complex that
Introduction ●
ensures proper degradation of faulty proteins
along with cell homeostasis
● Ubiquitin is a small regulatory protein that can
be added to protein complexes to mark for
degradation, alter in function, or change activity
levels
● Cells require functional proteasomes and
ubiquitin proteins in order to remain healthy and
prevent a variety of diseases

1
Glickman MH, Ciechanover A (April 2002). "The ubiquitin-proteasome
proteolytic pathway: destruction for the sake of construction". Physiological
Reviews. 82 (2): 373–428. doi:10.1152/physrev.00027.2001.

2
Schnell JD, Hicke L (September 2003). "Non-traditional functions of ubiquitin
and ubiquitin-binding proteins". The Journal of Biological Chemistry. 278 (38):
35857–60. doi:10.1074/jbc.R300018200.
 ● 26S is made of two sub-complexes:
○ Catalytic core particle (20S CP)

Organelle Structure ○ 1 or 2 Terminal 19S Regulatory Particles (19S

RP)
○ Composed of four axially stacked
heteroheptameric rings (two outer α- and two
inner β-rings)
○ Barrel-shaped structure
● 20S CP
○ Degrades the proteins
○ Capped at ends by regulatory proteins
○ ~750 kDa
● 19S RP
○ Binds to one or both ends of the 20S CP to form
an enzymatically active proteasome
○ Plays a role in recognizing, unfolding, and
translating proteins into 20S CP interior
○ ~700 kDa
3
Tanaka, Keiji. “The proteasome: overview of structure and functions.” Proceedings of
the Japan Academy. Series B, Physical and biological sciences vol. 85,1 (2009): 12-36.
doi:10.2183/pjab.85.12

4
Livneh, I., Cohen-Kaplan, V., Cohen-Rosenzweig, C. et al. The life cycle of the 26S
proteasome: from birth, through regulation and function, and onto its death. Cell Res
26, 869–885 (2016). https://doi.org/10.1038/cr.2016.86
 26S Proteasome is found in the nucleus and
Organelle Location ●
cytoplasm of all eukaryotic cells
● Large, approximately 2.5 MDa or 2,500 KDa
● 26S Proteasome performs ubiquitination
process in the cytoplasm
● Tight control of gene expression is used in
order to ensure proper balance of cell
proteasome levels
● Proteasomes can comprise ~1% of the total
protein amount in some mammalian cells

5
Marshall, Richard S., and Richard D. Vierstra. “Dynamic Regulation of the
26S Proteasome: From Synthesis to Degradation.” Frontiers in Molecular
Biosciences, vol. 6, 2019, doi:10.3389/fmolb.2019.00040.
Proteasome Function

● 2 different parts: 19S RP and 20S CP that have

different functions that work hand in hand to
degrade proteins.
○ 19S has two parts: Lid and Base.
3
Tanaka, Keiji. “The proteasome: overview of structure and functions.”
Proceedings of the Japan Academy. Series B, Physical and biological
sciences vol. 85,1 (2009): 12-36. doi:10.2183/pjab.85.12

4
Livneh, I., Cohen-Kaplan, V., Cohen-Rosenzweig, C. et al. The life cycle of
the 26S proteasome: from birth, through regulation and function, and onto its
death. Cell Res 26, 869–885 (2016). https://doi.org/10.1038/cr.2016.86
Deficiency of
Proteasome ● Functions:
○ Degrade damaged proteins:
■ Excessive apoptosis
○ Immune systems:
■ Generate antigenic peptides
■ Recognition of T-Cells
○ Replenish molecules:
■ Recycling of amino acids
● Deficiency:
1. Upregulation of apoptosis and cell
growth/metabolism
2. Missing antigenic peptides
3. Inefficiently making amino acids

6
Lecker, Stewart H, et al. “Protein Degradation by Ubiquitin-Proteasome Pathway
in Normal and Disease States.” American Society of Nephrology, 13 Apr. 2006, pp.
1807–1819., doi: 10.1681/ASN.2006010083.

7
Grammling, Josh. “Degredation of Ubiquitinated Proteins by 26S Proteasome”.
Grammling Medical Illustration, 13 Apr 2006.

Ubiquitin Structure
10
Vijay-Kumar S, Bugg CE, Cook WJ. Structure of ubiquitin
refined at 1.8 A resolution. J Mol Biol. 1987;194(3):531–
544. doi:10.1016/0022-2836(87)90679-6
Ubiquitin
● 87% hydrogen bonding in secondary structure
● Unique secondary structures
○ β-bulge, two reverse axis turns, and a
symmetrical hydrogen-bonding
Ubiquitin-protein ligase E3A
● General L shape
● Two lobes
○ Amino-terminal and carboxyl-terminal
● Ubiquitination
○ Thioester between cysteine and carboxyl
terminal
8
Tomaić, V, and L Banks. “Angelman Syndrome-Associated Ubiquitin Ligase
UBE3A/E6AP Mutants Interfere with the Proteolytic Activity of the Proteasome.”
Cell Death & Disease, vol. 6, no. 1, 2015, doi:10.1038/cddis.2014.572.
9
Huibregtse, J. M., et al. “A Family of Proteins Structurally and Functionally
Related to the E6-AP Ubiquitin-Protein Ligase.” Proceedings of the National
Academy of Sciences, vol. 92, no. 11, 1995, pp. 5249–5249.,
doi:10.1073/pnas.92.11.5249-a.
Protein Location ● Cytoplasm
○ Degradation, change of
location
● Nucleus
○ DNA repair, replication,
transcription and quality
control
● Cell surface membrane
○ Degradation and endocytosis

10
Vijay-Kumar S, Bugg CE, Cook WJ. Structure of ubiquitin
refined at 1.8 A resolution. J Mol Biol. 1987;194(3):531–544.
doi:10.1016/0022-2836(87)90679-6

11
Mikecz, A. Von. “The Nuclear Ubiquitin-Proteasome System.”
Journal of Cell Science, vol. 119, no. 10, 2006, pp. 1977–1984.,
doi:10.1242/jcs.03008.
 Signaling mechanism for proteasome
Protein Function ●
● Involves three ubiquitin activating enzymes
1. E1: activates ubiquitin w/ ATP
2. E2: transfers ubiquitin to E3
3. E3: transfers ubiquitin to target protein
● Forms isopeptide bond
○ C terminus of ubiquitin and ε-amino
group of lysyl group of target protein

Welchman, Rebecca L., et al. “Ubiquitin and

12

Ubiquitin-like Proteins as Multifunctional Signals.”

Nature Reviews Molecular Cell Biology, vol. 6, no. 8,
Aug. 2005, pp. 599–609., doi:10.1038/nrm1700.

13
Welchman, Rebecca L., et al. “Ubiquitin conjugation
and the ubiquitin–proteasome system.” Nature Reviews
Molecular Cell Biology, vol. 6, no. 8, Aug. 2005, pp.
599–609., doi:10.1038/nrm1700.
 ● 26S Proteasome is comprised of two subunits: 20S
CP and 19S RP

Summary ● 26S Proteasome is located in the cytoplasm and

nucleus of all eukaryotic cells
● The proteasome is a highly sophisticated protease
complex designed to carry out selective, efficient
and processive hydrolysis of client proteins.
○ It is known to collaborate with ubiquitin,
which polymerizes to form a marker for
regulated proteolysis in eukaryotic cells.
● A deficiency in proteasome leads to:
1. Unnecessary apoptosis
2. Lack of antigens peptide
3. Reusable amino acids
● Ubiquitin is found ubiquitously and marks proteins
for degradation or movement.
● Ubiquitination involves three ubiquitin enzymes for
regulation