4. Autophagy

​

Autophagy is a synchronized machinery, which results in the degradation of unuseful cellular constituents (Mizushima N and Komatsu M., 2011) (Dyshlovoy S A., 2020). 

 

A lysosome system is responsible for the degradation (selective or-non-selective) of proteins and other cell components produced by autophagy and non-selective degradation can mainly be initiated by cellular stress, such as starvation and toxins. This, in turn, leads to degradation and reutilisation of confiscated substances as a result of foundation of double-membrane vesicles (autophagosome), which afterward combines with lysosome (Yang Z J et al., 2011) (Mathew R et al., 2007) (Dyshlovoy S A., 2020). 

 

The important stages of autophagy are as below (Dyshlovoy S A., 2020):

 

1. The activation of autophagy; which starts with the stimuli- instigation episode including starvation, radiation and drug treatment, etc (Dyshlovoy S A., 2020). 

2. The foundation of phagophore (a double membrane compound developing in the cytosol): at this stage, lipidation occurs as LC3-I changes to LC3-II through binding to phosphatidylethanolamine (PE).

3. The foundation of autophagosome: Autophagosomes are vesicles, which are developed as phagophore vaguely surrounds the major part of the cytoplasm, such as the whole organelles, or it aims precisely for the organelles contents.

4. Curbing and merging with lysosome: At this stage, autolysosome is developed as a result of merging the autophagosome with the lysosome.

5. Vesicle collapse and degradation: At this final stage the lysosome proteases breakdown the confiscated substances and organelles within the autolysosome. This, in turn, leads to production of the ATP and number of macromolecules like proteins by recycled materials. 

 

Autophagy Introduction References

 

1.        Dyshlovoy, S. A. Blue-Print Autophagy in 2020: A Critical Review. Mar. Drugs 18, (2020).

2.        Mathew, R., Karantza-Wadsworth, V. & White, E. Role of autophagy in cancer. Nat. Rev. Cancer 7, (2007).

3.        Mizushima, N. & Komatsu, M. Autophagy: Renovation of Cells and Tissues. Cell 147, 728–741 (2011).

4.        Yang, Z. J., Chee, C. E., Huang, S. & Sinicrope, F. A. The Role of Autophagy in Cancer: Therapeutic Implications. Mol. Cancer Ther. 10, (2011).

​

4.1. Molecular Mechanism of Autophagy

 

A number of incidences in the organism, such as starvation and hypoxia leads to the activation of autophagy (Ichimiya T et al., 2020). 

 

Autophagy initiation results in recruitment of Unc-51 Like Autophagy Activated Kinase 1 (ULK1)/autophagy-related gene (Atg) 1 complex, including autophagy-related factors (Ichimiya T et al., 2020). This leads to the development of a membrane vesicle, phagophore and consequently autophagosome, which is a spherical lipid bilayer vesicle that combines with a lysosome or a vacuole in order to degrade unwanted cytoplasmic substances that is occupied within itself (Mizushima N and Komatsu M., 2011) (Ichimiya T et al., 2020).

 

One of the most critical controlling factors of the autophagy-lysosomal pathway is the Transcription Factor EB (TFEB). TFEB initiates the expression of autophagy and lysosomal genes as it controls the starvation-activating autophagy transcriptional regulation (Settembre C et al., 2011) (Ichimiya T et al., 2020). 

 

As starvation prevails, then the Mammalian Target of Rapamycin Complex 1 (mTORC1) kinase complex is inhibited, TFEB phosphorylation is suppressed and TFEB is translocated into the nucleus, where it attaches to the Coordinated Lysosomal Expression And Regulation (CLEAR) region of the gene and activates gene expression responsible for the progression of autophagy and lysosomal exocytosis (Palmieri M et al., 2011) (Setttembre C et al., 2012) (Napolitano G et al., 2018) (Ichimiya T et al., 2020).

 

The mTORC1 kinase complex (a serine/threonine kinase) belongs to family class III PI3K and it is classed as an inhibitor of autophagy (Saxton R A and Sabatini D M., 2017) (Ichimiya T et al., 2020). 

 

The mTORC1 kinase complex is also a negative regulator of the ULK1 complex as it phosphorylates ULK1 and Atg13. This negative regulation activity of mTORC1  consists of four features: (i) ULK1, (ii) Atg13, (iii) Focal adhesion kinase family Interacting Protein (FIP) 200 and (iv) Atg101 (Kim J et al., 2011) (Ichimiya T et al., 2020). Furthermore, mTORC1 through managing the TFEB localisation to lysosomes, controls autophagy (Roczniak –Ferguson A et al., 2012) (Ichimiya T et al., 2020). Also, mTORC1 is induced through the establishment of the GTP bound state of the brain enriched low molecular weight G protein Ras homology (Rheb), once it is on the exterior of lysosomes (Yang H et al., 2017) (Ichimiya T et al., 2020).

 

Rag (a low molecular weight G protein) controls the subcellular localisation of mTORC1. When amino acid starvation happens, then the conformation and activity of mTORC1 changes, which is a result of attaching to GDP/GTP through Rag A/B and Rag C/D heterodimers contained by Rag (Takahara T et al., 2020) (Ichimiya T et al., 2020).

 

Furthermore, phagophore is developed as a result of inhibition of mTORC1 and assembling a PI3K complex downstream of ULK1 (Ichimiya T et al., 2020). A number of complexes with various constituent features such as the complex containing Vacuolar Protein Sorting (Vas) 34, VAS 15, Beclin-1 and Atg14, which play a role at the initial steps of phagophore development, are part of class III PI3K complex. The Phosphatidylinositol-3-phosphate (PI3P) is known to be formed by VPS34 (a part of PI3K complex) (Boukhalfa A et al., 2020) (Ichimiya T et al., 2020). Additionally, WIPI2 can take action on Atg2 and WD repeat domain is known as a phosphoinositide interacting protein (WIP1) and is organised as a PI3P effector protein  (Ichimiya T et al., 2020). Phagophore and Endoplasmic Reticulum (ER) are joined by Atg2, which can carry lipid and is responsible for providing lipids to autophagosomes (Osawa T and Noda N N., 2019) (Ichimiya T et al., 2020). 

 

Autophagosomes are developed by two kinds of ubiquitin-like binding reaction systems i.e., the microtubule-associated protein light chain 3 (LC3) binding reaction system and Atg12-Atg5 binding reaction system (Ichimiya T et al., 2020). 

 

As LC3 joins the Phosphatidyl Ethanolamine (PE), then it causes the stretching and closure in the autophagosome membrane (Ichimiya T et al., 2020). Additionally, Atg 7 and Atg10 are responsible for the covalent binding of Atg12 and Atg5, which then results in the Atg12-Atg5 binding system relating and attaching to Atg16L1 (Lystad A H et al., 2019) (Cadwell K et al., 2008) (Ichimiya T et al., 2020).  In order for E3-like functionality regarding the LC3-PE complex develops, then the aforementioned conjugate needs to be assembled within the phagophore. The LC3, which is lipidised and encompasses E3-like functionality, can interrelate with a number of selective autophagy receptors and specifically destroy explicit substrates (Brier L W et al., 2019) (Galluzzi L and Green D R., 2019) (Ichimiya T et al., 2020).

 

As the phagophore stretched and achieved closure, then it results in the autophagosome reaching its finishing point (Ichimiya T et al., 2020). One of the factors that play a role in autophagosome closure is Endosomal Sorting Complex Required for Transport (ESCRT) complex. In order for the fusion of autophagosomes with lysosomes takes place, which in turn causes the development of autolysosomes that destroys the cargo, then there is a need for configuration of conjugates, synaptosomal-associated protein (SNAP) 29, lysosomal vesicle-associated membrane protein (VAMP) 7/VAMP8 and SNARE by two complexes i.e. soluble N-ethyl maleimide-sensitive protein (NSF) attachment protein receptor (SNARE) complexes (Takáts S et al., 2013) (Matsui T et al., 2018) (Ichimiya T et al., 2020).

 

Molecular Mechanisms of Autophagy References

 

1.        Brier, L. W. et al. Regulation of LC3 lipidation by the autophagy-specific class III phosphatidylinositol-3 kinase complex. Mol. Biol. Cell 30, (2019).

2.        Cadwell, K. et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 456, (2008).

3.        Galluzzi, L. & Green, D. R. Autophagy-Independent Functions of the Autophagy Machinery. Cell 177, (2019).

4.        Ichimiya, T. et al. Autophagy and Autophagy-Related Diseases: A Review. Int. J. Mol. Sci. 21, (2020).

5.        Kim, J., Kundu, M., Viollet, B. & Guan, K.-L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, (2011).

6.        Matsui, T. et al. Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17. J. Cell Biol. 217, (2018).

7.        Mizushima, N. & Komatsu, M. Autophagy: Renovation of Cells and Tissues. Cell 147, (2011).

8.        Napolitano, G. et al. mTOR-dependent phosphorylation controls TFEB nuclear export. Nat. Commun. 9, (2018).

9.        Osawa, T. & Noda, N. N. Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis. Protein Sci. 28, (2019).

10.     Palmieri, M. et al. Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways. Hum. Mol. Genet. 20, 3852–3866 (2011).

11.     Roczniak-Ferguson, A. et al. The Transcription Factor TFEB Links mTORC1 Signaling to Transcriptional Control of Lysosome Homeostasis. Sci. Signal. 5, (2012).

12.     Saxton, R. A. & Sabatini, D. M. mTOR Signaling in Growth, Metabolism, and Disease. Cell 168, (2017).

13.     Settembre, C. et al. TFEB Links Autophagy to Lysosomal Biogenesis. Science (80-. ). 332, (2011).

14.     Settembre, C. et al. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J. 31, (2012).

15.     Takahara, T., Amemiya, Y., Sugiyama, R., Maki, M. & Shibata, H. Amino acid-dependent control of mTORC1 signaling: a variety of regulatory modes. J. Biomed. Sci. 27, (2020).

16.     Takáts, S. et al. Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila. J. Cell Biol. 201, (2013).

17.     Yang, H. et al. Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40. Nature 552, (2017).

​

4.2. Mitophagy

 

As mitochondria are damaged, then they are selectively destroyed through mitophagy (Ichimiya T et al., 2020).

 

Mitochondria are involved in the production of ATP, synthesis of phospholipid and activation of apoptosis (Nunnari J and Suomalainen A., 2012) (Spinelli J B and Haigis M C., 2018) (Ichimiya T et al., 2020).

 

Outflow of electrons from the electron transport chain results in creation of Ros by mitochodria, which in turn can react with oxygen in the adjacent milieu (Nunnari J and Suomalainen A., 2012) (Ichimiya T et al., 2020).  As anomalous mitochondria are eliminated, then tumor development is repressed, which is achieved through mitophagy role in this process (Ichimiya T et al., 2020).

 

The deficit in Bcl-2 adenovirus EIB 19kDa-interacting protein 3 (BNip3), a hypoxia-activating protein (aiming at mitochondria), causes the loss of mitophagy, which in turn plays a role in an augmented development of mammary tumor (Chourasia A H et al., 2015) (Ichimiya T et al., 2020).

 

It is also identified that malfunctioning type of the phosphatase and tensin homolog (PTEN)-induced kinase (PINK)1/parkin complex is responsible for occurrence of hereditary Parkinson’s disease (PD).  Furthermore, this complex plays a role in phosphorylation of ubiquitin and throughout mitophagy functions as mitochondrial recognition marker (Scorziello A et al., 2020) (Ichimiya T et al., 2020).

 

Mitophagy References

 

1.        Chourasia, A. H. et al. Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis. EMBO Rep. 16, (2015).

2.        Ichimiya, T. et al. Autophagy and Autophagy-Related Diseases: A Review. Int. J. Mol. Sci. 21, (2020).

3.        Nunnari, J. & Suomalainen, A. Mitochondria: In Sickness and in Health. Cell 148, (2012).

4.        Scorziello, A. et al. Mitochondrial Homeostasis and Signaling in Parkinson’s Disease. Front. Aging Neurosci.12, (2020).

5.        Spinelli, J. B. & Haigis, M. C. The multifaceted contributions of mitochondria to cellular metabolism. Nat. Cell Biol. 20, (2018).