Caspase-independent signaling in programmed cell death

The family of caspases has long been regarded as the central executors of apoptosis and PCD in general. However, recent findings of evolutionary conserved forms of PCD, acting caspase-independently, extended the knowledge about intracellular signaling pathways.

1. Introduction

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Many non-caspase proteases are able to cleave classical caspase substrates and thus might mimic their cellular effects. The best examined non-caspase proteases in caspase-independent PCD are calpains, cathepsins and granzymes. These proteases can act either with caspases in a cooperative way or also completely independent from caspase activity to trigger and execute PCD often characterized by a typically apoptotic morphology ^72-76.

1.3.3.1 Calpain

Calpain is an ubiquitous cysteine protease with two major isoforms, m- and µ-calpain ^77,78. They reside inactive within the cytosol and are activated by intracellular elevation of Ca²⁺

concentration ^73,75,79. Activity may be further enhanced by proteolytic cleavage and association with membrane phospholipids, presumably by lowering the requirements of Ca²⁺. Calpain activity is controlled by calpastatin, a natural inhibitor, which is subjected to cleavage by calpain or caspases. Calpains share some common substrates with caspases, e.g. Bid, Bax and even procaspases-3 and -9 themselves ⁸⁰. However, cleavage of the latter two neither activates nor inactivates these enzymes ². Several stimuli like irradiation, etoposide or neurotoxins, which are able to enhance intracellular Ca²⁺-level induce calpain activity. Beside their cooperative activity within the classical caspase pathway several reports describe also caspase-independent apoptosis-like PCD induced by vitamin D analogues in breast cancer cells ^81,82.

1.3.3.2 Cathepsins

Until recently, the large family of lysosomal proteases was believed to be responsible for disposal of faulty proteins und for degradation of extracellular matrix once secreted.

However, more and more studies concede that they also have important roles in immunology and host defence as also in apoptosis, especially the cysteine proteases cathepsin B and L as well as the aspartate protease cathepsin D ^83-86.

Under special conditions, they have been found to be translocated from the lysosomes into the cytosol and nucleus before the appearance of morphological changes indicative for PCD.

Additionally, it was recognized that cathepsins are able to cleave caspases ^87-91. Cathepsins participate in caspase-dependent as well as caspase-independent PCD induced by TNF-a and by intrinsic stimuli like camptothecin, bile salts, oxidants and retinoids ^92-95. Interest in cathepsins was further boosted by the finding that the widely used caspase inhibitors zVAD-fmk, zDEVD-fmk and AcYVAD-cmk inhibited cathepsin B in an unspecific manner ^96,97.

1. Introduction

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Therefore, it has to be clarified in detail whether distinct phenomenon are indeed driven by cathepsins rather than by caspase activity.

1.3.3.3 Serine proteases

There is growing evidence that serine proteases, in combination with or completely independent of caspases, take part in protein degradation during apoptosis. Most of the evidence is based on the observation that particular apoptotic events can be prevented by broad-range inhibitorsof serine proteases such as TLCK or TPCK ^98,99.

Granzmye A and B are the most prominent effectors of PCD within this class of proteases.

They are stored in granules, which are utilized by cytotoxic T lymphocytes and natural killer cells for the elimination of pathogenic cells ². Upon activation, the contents of cytotoxic granules are released exocytotically to be delivered to target cells. The classical “lethal hit”

model stated thatfollowing granule exocytosis, poly-perforin formed channels in the target cell membrane in aCa²⁺-dependent manner through which granzymes A and B gained entry to the target cell ¹⁰⁰. Studies employing mice lacking granzyme B have demonstrated that this protease is required for the granule-induced rapid caspase-mediated apoptosis ⁷³. Granzyme B, like caspases,exhibits the unique feature of cleaving after aspartic acid residues and can thus directly activate the caspase cascade. Other substratescomprise the inhibitor of caspase-activated DNase (ICAD), poly ADP-ribose polymerase(PARP) and the proapoptotic BH3-only protein Bid ¹⁰¹. In the presence of caspase inhibitors, granzyme B triggers a slower, necrotic-like PCD ¹⁰². Granzyme A has a different substrate specifity as it cleaves its substrates after lysine or arginine residues. This protease induces death by activation of a caspase-independent granzyme A activated DNase leading to DNA single strand breaks ¹⁰³.

1.3.3.4 Mitochondrial effectors of caspase-independent PCD

Two caspase-independent proapoptotic factors are AIF and endonuclease G both translocating to the nucleus when released from mitochondria upon a cell death stimulus. They participate in DNA cleavage and chromatin condensation ^104-106. Whether these two proteins are released before, together or after cytochrom c release has been controversially discussed ¹⁰⁷. Endo G cleaves chromatin DNA into nucleosomal fragments independently of caspases. It is not exactly clear, how AIF contributes to large scale nuclear DNA fragmentation as it lacks intrinsic DNase activity, maybe it acts as a scaffolding protein to DNA for a yet unknown nuclease ¹⁰⁴.

1. Introduction

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The serine protease Omi/HtrA2 is also released from mitochondrial intermembrane space into the cytosol and the nucleus. The mature 36 kDa form can both neutralize and inactivate XIAP by either binding in a manner similar to Smac/DIABLO or by cleavage, thus potentiating apoptosis ^108-113. However, Omi/HtrA2 can also act caspase-independently as effector molecule in a necrosis-like PCD, which exclusively relies on its protease function^114-116.

Fig.1.4: Control of mitochondrial outer membrane permeabilization (MOMP) and downstream signaling after MOMP (adapted from Jäättelä ⁴).

In summary, it can be stated that the dependence of PCD in its diverse phenotypes on certain non-caspases, acting in parallel or cooperatively with caspase, might be extremely dependent on cell type and stimulus.