Why is lysosome called sucidal bag

Recycling the cell: autophagocytosis and the ubiquitin proteasome processes

Added by: Dr. Balint Földesi 02/04/19 8:00 am

Review article by Paul Domanski

For the regulated course of cellular functions, few factors are as decisive as the balance between the structure or synthesis and the breakdown of cytoplasmic organelles and proteins. The cell regulates this balance primarily via two mechanisms: 1) the ubiquitin-proteasome system and 2) autophagocytosis (self-eating ’), in which cellular components are fed to the lysosomal breakdown. While the ubiquitin-proteasome system primarily breaks down short-lived and misfolded proteins, the degradation of cytoplasmic components, damaged organelles and long-lived and aggregated proteins is carried out by means of autophagocytosis. This cellular ‘recycling’ system is highly regulated and thus enables the efficient recycling and regeneration of the cell components.

Ubiquitin

Ubiquitination, one of the most common post-translational modifications (PTM) in the cell, is the mechanism by which the majority of short-lived proteins are marked for degradation and breakdown. This is a highly specific system in which ubiquitin, a rather small protein with 76 amino acids, is linked to an epsilon amine of a lysine of the target protein via an amide bond. This is done using a three-step enzymatic process.

  1. Activation of ubiquitin by E1 (ATP-dependent).
  2. Transfer of the ubiquitin from E1 to the cysteine ​​of the active center of the ubiquitin-conjugating (UBC) enzyme E2 (there are more than 30 different E2 proteins).
  3. Ligation of the C-terminal glycine of ubiquitin to the target protein by one of the more than 500 E3 ubiquitin ligases.

Figure 1. The target proteins of the ubiquitin-proteasome system are ubiquitinated by E1, E2 and E3 using a three-step, ATP-dependent mechanism. The proteins are then unfolded in the proteasome and broken down into short peptide chains which can then be used in the context of the antigen presentation via MHC I or else hydrolyzed into the individual amino acids.

As part of the hierarchical ubiquitination cascade, E1 can bind dozens of E2 proteins, which in turn have the opportunity to associate with hundreds of E3 proteins. In addition, proteins can be monoubiquitinated, multiubiquitinated and polyubiquitinated. This increases the variability of the degradation system, since different ubiquitination patterns can activate different signaling pathways via binding to the more than 20 known specific ubiquitin binding domains. Similar to phosphorylation, ubiquitination is also reversible and ∼85 deubiquitinases (DUBs) have been described. As explained above, the majority of ubiquitinated proteins are broken down, but the modification with ubiquitin can also act as a transport signal for some proteins or mediate protein-protein interactions (Illustration 1).1-3

Ubiquitination is involved in various cell processes and malfunctions, including the immune response, DNA repair, signal transduction, cancer, neuropathies and many more. This naturally means that errors in the ubiquitination pathway have far-reaching and diverse effects in the affected cell and are thus causally involved in the development of a large number of neurodegenerative, cardiovascular and tumor diseases.4,5 As part of the great chemical and technological advances (e.g. mass spectrometry) of the last two decades, more and more active ingredients are now being developed that intervene in the ubiquitin-proteasome system.

Cayman Chemical offers you a wide range of products for researching the role and function of ubiquitin and the proteasome. One example are various inhibitors and agonists of ubiquitination, which enable the targeted analysis of individual processes or enzymes in the ubiquitin-proteasome system. In addition, Cayman offers you a range of antibodies, including FK1 and FK2, with which you can differentiate between specific ubiquitination patterns on the target proteins (monoubiquitination, multiubiquitination and polyubiquitination). Cayman Chemical recently added kits for ubiquitination detection, quantification of ubiquitin-binding proteins or deubiquitinase activity to its portfolio, which are essential tools for scientists in further researching the system.

Autophagocytosis

Autophagocytosis is an evolutionarily highly conserved cellular breakdown process in which cell components such as organelles are broken down by means of the lysosomal machinery. Consequently, autophagocytosis is a normal sub-process of cell growth, development, differentiation and homeostasis. Autophagocytosis enables the cell to maintain a delicate balance of synthesis, breakdown and recycling of cellular components. In lower organisms it regulates the metabolic balance when the supply of nutrients fluctuates.6 While it was originally assumed that autophagocytosis is a rather "crude" and poorly regulated process for the destruction of cell components, more recent findings have made it clear that autophagocytosis is also "selective". In higher organisms, this system eliminates aggregated proteins, damaged organelles such as mitochondria (mitophagia) or peroxisomes (pexophagia) and also eliminates microbial pathogens (xenophagia) (Figure 2).7,8 Although autophagocytosis takes place permanently in every cell, at least at a basal level, this activity can increase dramatically as a result of external and internal stimuli. Various signal transduction pathways mediate this stringent regulation, but the exact mechanisms behind it are currently often only incompletely investigated. A number of proteins involved are known, including mTOR, p62, Beclin 1 and PINK1 (Figure 3).8 Since autophagocytosis, just like ubiquitination, has to take place in constant balance with the new synthesis of proteins and organelles, it is to be expected that dysregulations of the system will have far-reaching, pathological consequences for the organism. In fact, it has been shown that knockout mice, which lack a gene that is crucial for autophagocytosis, die within hours of birth.9 It is also known that disorders in autophagocytosis play a role in cancer, neurodegenerative diseases (in both Huntington's disease and Alzheimer's disease there is an increased accumulation of autophagic vacuoles), lysosomal storage diseases, infectious diseases and the repair of DNA damage (over HDACs). It is also thought that autophagocytic malfunctions are involved in aging and cell death.10,11

Figure 2. Nonselective and selective autophagocytosis (A) Nonselective autophagocytosis is activated under hunger stress and mediates the breakdown of cytoplasmic components into usable smaller building blocks. (B) Stimuli such as the activation of p62, damage to mitochondria or infections can trigger the activation of selective autophagocytosis.

Autophagocytosis is primarily characterized by the presence of a unique organelle, the autophagosome. The autophagosome is a vacuole with a diameter of 300-900 nm, surrounded by a double membrane. It arises from the so-called phagophore, which expands and thereby includes cytoplasmic components. These phagosomes then fuse with the lysosome to form the active autolysosome.6

In the late 1990s, work on yeast led to the discovery of over 20 genes involved in autophagocytosis (ATGs). Further research eventually led to the description of the mammalian homologues of these genes and associated proteins.12,13 A schematic representation of the signal cascade and the known inhibitors and activators is in Figure 3 shown. In nutrient-rich conditions, the protein kinase mTOR acts as the primary negative regulator of autophagocytosis. Under starvation conditions, however, mTOR (the rapamycin target protein in mammals) is deactivated and thus ULK (the mammalian equivalent of the yeast protein ATG1) is activated, which then translocates into the endoplasmic reticulum and there initiates the autophagy cascade. The formation of the phagosome is completed by cleaving ATG8 (in mammals the light chain 3 of the microtubule-associated protein (MAP-LC3 or LC3-I)) C-terminally by ATG4 and coupling phosphatidylethanolamine (PE) to ATG8. This LC3 phospholipid (LC3-II) then binds covalently to the phagophore and is essential for the formation of the phagosome. For this reason, LC3-II can be used as a highly specific marker for the autophagosome. Just like ubiquitination, this process is reversible, and this delipidization is also mediated through ATG4.

Figure 3.The autophagous pathway in mammals (A) The prototypical induction of autophagocytosis by mTOR (inhibited by rapamycin) leads to (B) nucleation / expansion of the autophagosomal membrane with the help of a Beclin 1 and PI3K-containing protein complex (inhibited by 3-methyladenine (3-MA) ​​and wortmannin) . (C) The membrane is expanded by a mechanism similar to ubiquitination, in which ATG7 and ATG3 convert LC3-I to LC3-II, which in turn accumulates on the autophagosomal membrane.

Autophagous activity in cells can be quantified using a variety of methods. The first successful approaches included staining the autophagosomes with acidotropic dyes such as monodansylcadaverine (MDC) (Cayman’s Autophagy / Cytotoxicity Dual Staining Kit), as well as transmission electron microscopy (TEM).

Interestingly, the PE modification of LC3-I causes a change in the electrophoresis mobility, which enables the exact analysis of the LC3-I and LC3-II levels by Western blot. In combination with lysosomal inhibitors such as bafilomycin A1, this technique can be used to measure the accumulation of LC3-II in the autophagolysosome. This in turn allows the determination of the ‘autophagic flow’, i.e. the rate at which proteins and cell material are processed in the autophagosome. 14-17

The ubiquitin-proteasome system and autophagocytosis are of crucial importance for the delicate balance of production and degradation of proteins and organelles in all cells. Today it is known that these degradation pathways can also interact and that autophagocytosis under extreme stress also degrades ubiquitinated proteins. Although much has already been deciphered, some research is still necessary. Researchers around the world are working on identifying new biomarkers in order to better understand the functions of these systems and to develop innovative therapeutic options for the associated diseases.

credentials

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  2. Komander, D. and Rape, M. Annu. Rev. Biochem. 81: 203-209 (2012).
  3. Spasser, L. and Brik, A. Agnew. Chem. Int. Ed. Engl. 51 (28), 6840-6862 (2012).
  4. Fulda, S., Rajalingam, K., and Dikic, I. EMBO Mol. Med. 4 (7), 545-556 (2012).
  5. Shaid, S., Brandts, C.H., Serve, H., et al. Cell Death Differ. 20 (1), 21-30 (2013).
  6. Abeliovich, H. and Klionsky, D.J. Microbiol. Mol. Biol. Rev. 65 (3): 463-479 (2001).
  7. Jin, M., Liu, X., and Klionsky, D.J. Cell 152 (1-2), 368 (2013).
  8. Mizushima, N. and Komatsu, M. Cell 147 (4): 728-741 (2011).
  9. Komatsu, M., Waguri, S., Ueno, T., et al. J. Cell Biol. 169 (3): 425-434 (2005).
  10. Todde, V., Veenhuis, M., and van der Klei, I.J. Biochim. Biophys. Acta 1792 (1), 3-13 (2009).
  11. Robert, T., Vanoli, F., Chiolo, I., et al. Nature 471 (7336): 74-79 (2011).
  12. Klionsky, D.J., Cregg, J.M., Dunn, W.A., Jr., et al. Dev. Cell 5 (4): 539-545 (2003).
  13. Legakis, J.E. and Klionsky, D.J. Deretic, V., editor, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 1-17 (2006).
  14. Klionsky, D.J., Abdalla, F.C., Abeliovich, H., et al. Autophagy 8 (4), 445-544 (2012).
  15. Mizushima, N. and Yoshimori, T. Autophagy 3 (6): 542-545 (2007).
  16. Mizushima, N., Yoshimori, T., and Levine, B. Cell 140 (3), 313-326 (2010).
  17. Rubinsztein, D.C., Mariño, G., and Kroemer, G. Cell 146 (5), 682-695 (2011).

 

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