Proteasome function

Proteasome is a protein complex that plays a crucial role in maintaining cellular homeostasis by degrading misfolded and damaged protein substrates.

 

Key points about proteasome:

  1. Proteasome involved in the MHC class I-restricted antigen processing of ER-targeted proteins and also play a major role in processing of cytosolic proteins.
  2. Involved in the protection of cancer cells from tumor suppressor proteins and cytotoxic protein degradation.
  3. Involved in regulation of cell cycle by reducing the availability of cyclin proteins and other key factors, which are essential during cell division.
  4. Proteasomal mechanism linked to the Apoptosis. During apoptosis proteasomes degrade specific proteins, but on the other hand some components of the proteasome system are degraded by Apoptotic caspases (12).
  5. Involved in the biosynthesis and function of cellular organelles including mitochondria, chloroplast, ER, Golgi, lysosomes, centrosomes and ribosomes.
  6. Proteasome assist cell division by degrading the anaphase inhibitors.
  7. Regulates DNA transcription and DNA repair.
  8. Plays role in cell differentiation and development
  9. Involved in neural and muscular degeneration.
  10.  Regulates dynamic cellular mechanisms such as endocytosis, proteolysis, and immune signaling.
  11. Involved morphogenesis of neural networks
  12.  Involved in the inflammation and Immune responses.
  13. This protein plays a major role in modulation of cell surface receptors, ion channels, and the secretory pathway.
  14. Regulates bone remodeling mechanism.
  15. Involved in the inhibition of Osteoclastogenesis by degrading the RANKL, thus, inhibiting the osteoclastic bone resorption directly.
  16. Ubiquitin-proteasome machinery also regulates osteoblast bone formation by inhibiting the osteoblastogenesis.
  17. Response to stress and extracellular modulators.
  18.  Involved in ribosome biogenesis.
  19. Regulates viral infection by degrading the protein coat of virus.

 

1. Proteasome importance in the cell

Regulated protein hydrolysis in eukaryotic cells is crucial for cell viability through removal of highly toxic misfolded, damaged or unwanted proteins before they accumulate to reach toxic levels. This important molecular proteolytic machine also plays a pivotal role in regulation of cell cycle by controlling the concentrations of cyclin and other key proteins in a time-dependent manner. Proteasome machinery also plays role in immune response by generating antigenic peptides. In recent studies, surprisingly, proteasome complex machinery is considered as a major drug target to control certain types of cancer.

 

2. Discovery

Avram Hershko and his graduate student, Aaron Ciechanover and other scientist, Irwin Rose discovered the ubiquitin dependent protein degradation in in the late 1970s and early 1980s and awarded Nobel Prize jointly in Chemistry 2004 for the discovery of Ubiquitination.

 

3. Proteasome complex structure and function

Proteasomes are complex of multisubunit protease enzymes and have cylindrical shape. Proteasomes are found in both prokaryotes and eukaryotes and involved in ubiquitination process. In eukaryotes they are present in both cytosol and nucleus. Proteasomes of eukaryotes are two types such as 20S proteasome and 26S proteasome. The 20S proteasome is a cylindrical barrel-shape structure composition of 28 subunits, which are arranged as four rings of heptamers fixed upon one another.  These all subunits belong to same super family of proteins, however which are grouped into two families, represented as alpha and beta. In eukaryotes, 26S type proteasome is formed by assembled with two 19S regulatory proteins, which is ATP required process (see the figure).

Proteosome complex
Figure: The 26S proteasome is a protease enzyme complex found in the cytosol, perinuclear regions and nucleus of eukaryotic cells. 26S proteasome composite of 20S proteasome (28 subunits core) and two 19S regulatory proteins. 20S proteasome consist of two outer and two inner heptameric rings, which are stacked axially to form hallow cylindrical structure in which protein degradation takes place. 19S regulatory units composite of 19 individual proteins and forms cap shaped structure that mediates the entry of contempt protein.

The 26S proteasome digests proteins by using cellular energy, however, these proteasomes cannot degrade cellular proteins without proteolytic signals, which are generated from ubiquitination process. Ubiquitination-mediated proteolysis is regulated process, that involves various regulatory proteins. Ubiquitination mechanism produces polyubiquitinated protein (susceptible protein to degrade) that will be degraded by proteasome.

Proteosome complex

 

The reaction starts with attachment of ubiquitin to damaged protein. In first step, terminal carboxyl group of ubiquitin interact with E1 by thioester bond by using energy, which is released by ATP hydrolysis. In second step, this activated protein is subsequently transferred to a sulfhydryl group of E2. In final step, E3 catalyzes the separation of attached ubiquitin molecules from E2 and transfer it on target protein.

4. Function of 20S proteasome core

The 20S core particle of proteasome complex is a molecular machine that plays an important role in cellular function to degrade misfolded or unwanted protein substrates (which is nonlysosomal protein degradation), thereby ensuring protein homeostasis in eukaryotic cells.

 

5. Function of 19S regulatory units

19S regulatory unit (gate) has ATPase activity that regulates the proteasomal function through a gating mechanism that controls sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricts access to catalytic sites sequestered in the lumen of the structure. This unit mainly involved in three functions. First, this unit recognizes and binds specifically to polyubiquitin chains, thereby ensuring that only ubiquitinated protein to be degraded. Second function, this 19S unit removes intact molecules from the fated protein so that they can be reused. Third function, this 19S unit unfolds fated protein and sends that into catalytic lumen or core.

 

6. Proteasome as a target for cancer treatment

The continuous proliferation of cancer cells requires immediate turnover of expired or misfolded proteins, as well as the efficient degradation of tumor suppressor proteins and pro-apoptotic factors such as the p53 protein. Because, this immediate turnover provides free amino acids to reuse for synthesis of new proteins in cancer cells, while, efficient degradation of tumor suppressor and pro-apoptotic proteins protects the cancer cells from self-degradation.

Due to this advanced feature, cancer cells are more powerful than normal cells in order to control the cytotoxic effect, which is formed by accumulation of unwanted proteins.

 

7. Proteasome inhibitors

In earlier studies, scientists have recognized numerous small-molecule inhibitors of the proteasome complex. Especially, these Inhibitors related to the peptide aldehyde group that inhibit the proteasome function reversibly and are explored extensively in research. However, they can also inhibit the function of cellular proteases. Since, they have ability to control protease activities, they have been used as a research tools and as a potential drugs against various cancers.

 

Beta-lactone

Beta-lactone can inhibit proteasome by binding irreversibly to the active-site threonine of the β subunits in the 20s proteasome, but they are not completely proteasome-specific inhibitors.

Epoxyketone classes

Carfilzomib and Oprozomib are epoxyketone classes of proteasome inhibitors that are more specific for the proteasome and as such have successfully made the leap from research to therapeutic applications.

Dipeptidyl boronic acid class

The bortezomib is a dipeptidyl boronic acid class proteasome inhibitor that can inhibits the proteasome. The recent studies stated that application of bortezomib during bone formation induced the osteoblast differentiation by protecting differentiation-related transcription factors from proteasomal-mediated protein degradation.

Disulfiram

Disulfiram and its analogue pyrrolidine dithiocarbamate (PDTC) have been recognized as partial noncompetitive proteasome inhibitor that can bind slowly to 20S proteasome and inhibits catalytic activity. Hence, these are used against chronic lymphocytic leukemia (10, 16).

Marizomib

Marizomib (salinosporamide A) has used in clinical trials against multiple myeloma. This drug can inhibit all three proteasome subunits.

Other drugs

Delanzomib is also used in clinical trials against cancer.

Epoxomicin is a naturally occurring selective inhibitor for protein degradation.

Epigallocatechin-3-gallate has also been proposed as potent inhibitor.

Beta-hydroxy beta-methylbutyrate is a proteasome inhibitor used in human skeletal muscle.

 

References

  1. Banaszynski et al., “A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules,” Cell, 126:995-1004, 2006.
  2. Garrett IR1, Chen D, Gutierrez G, Zhao M, Escobedo A, Rossini G, Harris SE, Gallwitz W, Kim KB, Hu S, Crews CM, Mundy GR. Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro.J Clin Invest. 2003 Jun;111(11):1771-82.
  3. Gustafson et al., “Small molecule mediated degradation of the androgen receptor through hydrophobic tagging,” Angew Chem Int Ed Engl, 54:9659-62, 2015.
  4. Xie et al., “Pharmacological targeting of the pseudokinase Her3,”Nat Chem Biol, 10:1006-12, 2014.
  5. Uyama M1, Sato MM, Kawanami M, Tamura M. Regulation of osteoblastic differentiation by the proteasome inhibitor bortezomib.Genes Cells. 2012 Jul;17(7):548-58. doi: 10.1111/j.1365-2443.2012.01611.x. Epub 2012 Jun 15.
  6. Bondeson et al., “Catalytic in vivo protein knockdown by small-molecule PROTACs,” Nat Chem Biol, 11:611-17, 2015.
  7. Jeanmarie Verchot. Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum. Viruses. 2016 Nov; 8(11):
  8. Schneekloth et al., “Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics,” Bioorg Med Chem Lett, 18:5904-08, 2008.
  9. Winter et al., “Phthalimide conjugation as a strategy for in vivo target protein degradation,” Science, 348:1376-81, 2015.
  10. Lövborg H, Oberg F, Rickardson L, Gullbo J, Nygren P, Larsson R. Inhibition of proteasome activity, nuclear factor-KappaB translocation and cell survival by the antialcoholism drug disulfiram. Int J Cancer. 2006 Mar 15;118(6): 1577-80.
  11. Raina et al., “PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer,” PNAS, 113:7124-29, 2016.
  12. Wójcik C. Regulation of apoptosis by the ubiquitin and proteasome pathway. J Cell Mol Med. 2002 Jan-Mar;6(1): 25-48.
  13. Buckley et al., “HaloPROTACS: Use of small molecule PROTACs to Induce degradation of HaloTag fusion proteins” ACS Chem Biol, 10:1831-37, 2015.
  14. Orlowski RZ. The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ. 1999 Apr;6(4): 303-13.
  15. Hines et al., “Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs” PNAS, 110:8942-47, 2013.
  16. Hasinoff BB, Patel D. Disulfiram is a slow-binding partial noncompetitive inhibitor of 20S proteasome activity. Arch Biochem Biophys. 2017 Nov 1;633: 23-28. doi: 10.1016/j.abb.2017.09.003. Epub 2017 Sep 5.

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