Upstream eventDisruption, Lysosome
N/A, Mitochondrial dysfunction 1
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Endocytic lysosomal uptake leading to liver fibrosis||adjacent||High|
Life Stage Applicability
|All life stages|
Key Event Relationship Description
Disrupted lysosomal membrane release the content of lysosomes including cathepsins. Cathepsins take part in activation of BH3-only proteins, which directly or indirectly activate pro-apoptic Bax and Bak proteins. Once activated Bax and Bak form dimers and higher order oligomers, in order to form pores in outer mitochondrial membrane and cause mitochondrial injury.
Many evidences suggest that lysosomal disruption usually precedes mitochondrial injury (Guicciardi et al., 2000; Brunk et al., 2001; Zhao et al. 2003; Droga-Mazovec et al., 2008), with lysosomal proteases inducing mitochondrial dysfunction.
Zhao and colleagues have also proposed the existence of a positive feed-back mechanism between lysosomal damage and mitochondrial damage, in which early lysosomal rupture causes mitochondrial rupture and leakage of mitochondrial proteins that increase lysosomal damage and consequent apoptosis (Zhao et al., 2000).
The pathway between lysosomal membrane permeabilization (LMP) and mitochondrial membrane permeabilization (MMP) is regulated principally by Bcl-2 family of proteins. The family is subdivided into anti-apoptotic multidomain proteins (such as Bcl-2, Bcl-xl, Bcl-W, Mcl-1 and A1), pro-apoptotic multidomain proteins (Bax and Bak) and pro-apoptotic BH3-only proteins (such as Bid, Puma, Noxa, Bim, Bad, and Bik) (Fletcher and Huang, 2006; Youle and Strasser, 2008).
Cathepsins, released after lysosomal damage, have a role in the cell death through the cleavage of BH3-only proteins, such as Bid, to generate active tBid (truncated Bid) (Blomgran et al., 2007; Cirman et al., 2004; Droga-Mazovec et al., 2008; Houseweart et al., 2003; Stoka et al., 2001) and by degradation of the anti-apoptotic Bcl-2 molecules Bcl-2, Bcl-xl and Mcl-1 (Blomgran et al., 2007; Droga-Mazovec et al., 2008). However it was shown that though Bid is not the only substrate of lysosomal enzymes that induce cytochrome c release, it is the major one (Stoka et al., 2001). Droga-Mazovec and colleagues showed that Bid is cleaved by cathepsins in human liver carcinoma cells (HepG2) (Droga-Mazovec et al., 2008), while other study showed that particularly cathepsin B is active in hepatocytes (Guicciardi et al., 2000).
Bid is also cleaved by caspase 8, which represents a link between extrinsic and intrinsic (mitochondrial) pathway (Li et al., 1998).
Activated BH3-only proteins continue to activate pro-apoptic proteins Bax and Bak. Sarosiek et al. observed that Bid preferentially activates Bak, while Bim activates Bax (Sarosiek et al., 2013). The activation of Bax and Bak occurs after LMP, but before mitochondrial release of cytochrome c and caspase-3 activation (Boya et al., 2003). Currently there are two models describing activation of Bax and Bak proteins and the role of anti-apoptic and pro-apoptic multidomain proteins in it. In the indirect model, Bax and Bak are sequestered and inactivated by anti-apoptotic Bcl-2 proteins. The binding of pro-apoptotic BH3-only proteins to these Bcl-2 proteins triggers the release of Bax and Bak. The direct model proposes that Bax and Bak are activated by direct binding of pro-apoptotic BH3-only proteins, called the activators (Bid, Bim or Puma). However, these activators are normally sequestered by anti-apoptotic Bcl-2 proteins. In order to release the activators, other BH3-only proteins, called senzitizers, neutralize anti-apoptotic Bcl-2 proteins (Brenner and Mak, 2009; Willis et al., 2007).
Bak and Bax go under major conformational changes after binding of BH3 only proteins (as reviewed by Westphal et al., 2014). Once activated Bak or Bax molecules bind reciprocally to form symmetric homodimers. It is thought that homodimers of Bak or Bax must then associate to higher order oligomers to porate the mitochondrial outer membrane (Uren et al., 2017). Heterodimers form only a minor population compared with homodimers (Dewson et al., 2012; Mikhailov et al., 2003).
Bak and Bax shallow insertion into the outer leaflet of mitochondrial membrane (Westphal et al., 2014; Oh KJ et al., 2010) may destabilize the lamellar structure of the bilayer to induce lipidic pores in mitochondrial membrane. This induces release of proteins from the space between inner and outer mitochondrial membrane (Newmeyer et al., 2003).
Evidence Supporting this KER
In the last decade there is a growing body of evidences about the strong functional link between lysosomes and mitochondria that play an important role in physiology and pathology. The evidences also showed link between lysosomal and mitochondrial damage, and that lysosomal damage precedes mitochondrial injury.
LMP after exposure to ciprofloxacine, norfloxacine and hydroxychloroquine is detected couple of hourse earlier that MMP (Boya et al., 2003). The same study proves that the cells with signs of MMP are sub-ensemple of the group of the cells with signs of LMP, not vice-versa. Also inhibition of LMP with Baf A1 – that inhibits lysosomal vacuolar H+ ATP-ase, prevented MMP, while inhibition of MMP in Bax/Bak double knocks out cells didn't prevent LMP. However, inhibition of MMP prevented LMP to cause manifestations of the cell death. All the evidences from this and other studies (such as Droga-Mazovec et al., 2008; Ghosh et al., 2011) prove that LMP lies upstream from MMP and causes it.
When isolated mitochondria are incubated with purified cathepsin B in the presence of cytolic extracts, a release of cytochrome c from mitochondria is detected (Guicciardi et al., 2000). The microinjection of cathepsin D to the cell causes cytochrome c release, capsases activation and apoptosis (Roberg et al., 2002).
It was shown that mice deficient of stefin B (major intracellular cathepsins inhibitor) developed spontaneous cerebellar apoptosis (Houseweart et al., 2003). Pepstatin A, an inhibitor of cathepsin D, was found to inhibit caspase-3-like proteolytic activity and to prevent apoptosis (Roberg et al., 1999). The treatment of the cells with cathepsin B and cathepsin D inhibitors, pepstatin A and E-64-d, decreased MMP and activation of caspases (Kagedal et al., 2001).
Cathepsin B directly cuts Bid and produces active tBid, while cathepsin B inhibitors z-FA-fmk and E-64-d block Bid activation in cells (Zhang et al., 2009; Blomgran et al., 2007). When cathepsin B silenced HeLa cells were treated with granulysin Bid degradation was blocked, same as cytochrome c and apoptosis inducing factor (AIF) release (Zhang et al., 2009).
Stoka et al. showed that incubation of rat mitochondria to uncleaved Bid resulted only in insignificant levels of cytochrome c release, while exposure to both uncleaved Bid and lysosomal extracts resulted in cytochrome c release (Stoka et al., 2001). Other studies confirmed necessity of tBid for cytochrome c release (Gross et al., 1999; Luo et al., 1998).
Bid knockout mouse embryonic fibroblasts (MEFs) and Bax/Bak deficient MEFs were more resistant to granulysin induced death compared to wild type, and they released less cytochrome c and AIF (Zhang et al., 2009; Lindsten et al., 2000). Bcl-2-overexpressed HeLa cells almost completely blocked the release of cytochrome c and AIF after granulysin treatment (Zhang et al., 2009). Overexpression of Bcl-2 is also suppressing oxidative stress induced apoptosis (Zhao et al., 2000).
Some studies stated that certain cell types such as hepatocytes appear to require a Bid in order to disrupt mitochondrial membrane, release cytochrome c and following steps to execute apoptosis (Korsmeyer et al., 2000). They also proved that BH3 domain of tBid was not required for targeting mitochondrial membrane but it is required for cytochrome c release.
Using specific inhibitors it was demonstrated that cytosolic cathepsin D triggers Bax activation and translocation to mitochondria, resulting release of AIF from mitochondria, and the apoptosis (Bidere et al., 2003).
Incubation of cleaved Bid with mitochondria promotes oligomerization of membrane bound Bak and cytochrome c release (Wei et al., 2000).
Bax/Bak double knockout MEFs didn't show MMP induced by ciprofloxacin, norfloxacin and hydroxychloroquine, while increased LMP was detected (Boya et al., 2003).
Incubation of Bax with isolated mitochondria resulted in cytochrome c release while Bcl-xl inhibits this release (Jurgensmeier et al., 1998)
The length of the core dimer of Bax or Bak is the approximate width of the mitochondrial outer membrane, and the bend observed in the structures may be accommodated by the curved edge of the pore (Uren et al., 2017).
Uncertainties and Inconsistencies
Repnik and colleagues showed that inhibition of cysteine cathepsins by E-64-d had little effect on LDH (cytosolic enzyme lactate dehydrogenase) release in medium in LLOMe treated cells (Repnik et al., 2017). They also detected that after exposure to LLOMe, cathepsins remain in lysosomes and are being degraded there which is in contradiction with most of the previous studies.
As stated earlier there are empirical evidences that incubation of cathepsin B with mitochondria and cytosolic factors increase mitochondrial permeabilization. However, in some studies pharmacological inhibition of cathepsin B, L and D didn't suppress Bid cleavage, suggesting that other lysosomal proteases might be responsible for Bid cleavage (Reiners et al., 2002).
The knockout of genes coding for cathepsins B, D, L and S failed to prevent induced MMP and cell death (Boya et al., 2003).
Housewert et al. showed that the amount of cerebellar granule cell apoptosis in cystatin B-deficient mice did not change when Bid was removed. This indicates that cathepsins can use other mechanisms to initiate apoptosis. They concluded that another molecule may partially substitute for Bid when it is missing (Housewert et al., 2003). Willis and colleagues showed that neither Bim nor Bid are necessary for apoptosis, as their absences didn't stop apoptosis or prevented Bax activation (Willis et al., 2007).
Some reports described Bax/Bak-independent mechanisms of cytochrome c release, involving either cyclosporine A sensitive mitochondrial membrane permeability (Wan et al., 2008) or a serine protease(s)-dependent mechanism (Mizuta et al., 2007).
Quantitative Understanding of the Linkage
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Murine (Stoka et al., 2001; Zhang et al., 2009; Lindsten et al., 2000)
Human (Boya et al., 2003; Cirman et al., 2004)
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