**3. Programmed cell death‐related proteases**

**1. Introduction**

26 Enzyme Inhibitors and Activators

signals [2].

Every proteinaceous enzyme that performs hydrolysis of peptide bonds is correctly designed as protease (peptidase) [1, 2]. This term was first used by Vines [3] based on direct and indirect evidences from studies with algae, some fungi and Phanerogams. Long before this work, the word "proteolytic" was applied by Roberts [4] to describe the digestive process on human stomach, and in the first years of the twentieth century, the digestive proteins trypsin and pepsin as well as other autocatalytic enzymes were known as proteases, being pepsin credited to do "proteolysis" since 1877 [5]. In 1928, Grassmann and Dyckerhoff [6] established important definitions concerning the nature of the catalytic activities of proteases. In resume, the peptide cleavage pattern by proteases can be internal, for endopeptidases; on the N‐terminal

Despite this difference, all proteases share the same catalytic strategy, as they polarize the carbonyl group on the peptide bond of the substrate by the stabilization of the oxygen atom on an oxyanionic hole, what makes the carbon atom more vulnerable to the attack of an activated nucleophile. The nature of the nucleophile presents some variation and is determinant to the employed mechanism for enzymatic catalysis. As a result of these variations, the proteases are grouped into six different catalytic families: serine, threonine, cysteine, aspartic, glutamic and metallopeptidases (http://merops.sanger.ac.uk/). The larger families (cysteine, serine, aspartic and metallopeptidases) have a wide range of distribution on living organisms, and are also

About their function, these enzymes are well known for the promotion of protein degradation on amino acid unities. Besides this function, they regulate the destination of other proteins based on their cleavage specificity and participate on important cellular pathways, being key regulators in different response processes to environmental factors and developmental

One of these cellular pathways is the event of programmed cell death (PCD) that, since the 2000s, is being studied with increased interest on non‐metazoan organisms, in particular on plant models. On metazoan, one of the main constituent of the pathway are cysteine proteases known as caspases. They are not found in plants, but an increasing number of reports shows strong evidences that caspase‐related proteases that belong to the metacaspase group, as well as proteases

Programmed cell death (PCD) is a genetically controlled physiological innate mechanism, which involves the selective death of individual cells, tissues or entire organs. It is a process different from necrosis as it occurs passively in response to environmental perturbations [9]. Together with the chromatin remodelling machinery, the cell cycle regulation mechanisms, the nuclear envelope and the cytoskeleton, this process is one of the major eukaryotic innova-

tive aspects, which allowed the development of more complex organisms [10].

portion, for aminopeptidases; or C‐terminal, for carboxypeptidases [2].

present in the "controversial" group of viruses [7].

with caspase‐like activities are eminent on plant PCD [8].

**2. Programmed cell death in plants**

Concerning to PCD, the main group of proteases performing important roles is the subfamily C14 from the CD clan of cysteine proteases. Their representatives include the metacaspases, paracaspases and caspases.

The first discovered and the most known in terms of structure and function are the caspases. The early reports of caspases and related proteins are those with the genes *cd‐3* and *cd‐4*, as well as their encoded products from *Caenorhabditis elegans* and the enzyme caspase‐1 from mammals [23–27].

To date, caspases have been proven to be "in the heart" of a pathway that mediates the highly ordered process of apoptotic cell suicide [28], and, indeed, they are the convergence point of biochemical pathways on cellular substrates which lead to the activation of a "protease cascade" (the caspase cascade).

#### 28 Enzyme Inhibitors and Activators


**Figure 1.** Representation of caspase, metacaspase and paracaspase major domains. IC: initiator caspase; EC: executor caspase; MI: type I metacaspase; MII: type II metacaspase; PC: paracaspase. Red: p20 domain; blue: p10 domain; orange: prodomain/recruitment domain; purple: death domain; green: immunoglobulin‐like domain; yellow: paracaspase C‐terminal region. Based on Vercammen et al. [8].

The importance of this cascade can be seen when disturbance of its regulation occurs on the cells, what causes immunodeficiency, carcinogenesis and other troubles related to aberrant PCD [29–31].

The cascade overview reveals two functional groups of caspases, concerning their position on the sequence of proteolytic events (**Figure 1**). The first caspases to be activated are the initiators which become active by the "induced proximity mechanism", often triggered by recruitment of adaptor protein complexes, such as recruitment domain of membrane receptors or by a huge protein complex called apoptosome. This induced proximity leads to the oligomerization of caspase molecules which form a heterodimer that becomes able to trigger the activation of the effector caspases by cleavage of a pro‐domain, since these enzymes are synthetized as zymogens. After this cleavage, the effectors themselves also stay organized as heterodimer, and cleave the apoptotic substrates, leading to a typical cellular morphology [8].

Despite the positional and functional differences, all caspases belong to a group named ICE family (interleukin‐1β‐converting enzyme/caspase 1 family), since caspase 1 was the first characterized member.

About the phylogeny, these molecules are unique and well distributed among the Animalia Kingdom. Their presence ranges from vertebrate organisms such as *Homo sapiens* and *Danio rerio* to invertebrates such *C. elegans* and *Drosophila melanogaster* [32]. Also, their involvement with cell death processes is also well documented.

Alongside caspases, on clan CD, there are caspase relatives called paracaspases which are found in metazoans and in slime molds [4, 32] (**Figure 1**). In humans, paracaspase/MALT1 is associated to the lymphocyte activation by the NF‐kB pathway [33, 34]. In *Dictyostelium*, the paracaspase gene disruption does not affect the developmental cell death process, what leads to the suggestion that the enzyme is not required for the phenomenon [35]. A function for human paracaspase MALT 1 on caspase‐8 modulation is reinforced by Kawadler et al. [36], whose data show that MALT1 activates caspase‐8 during TCR signalling but reduces the cleavage of caspase‐3, avoiding apoptosis and inducing cell proliferation.

About its structural architecture, the human MALT 1 monomer has an apparent molecular mass of 41 kDa and the dimer, about 84 kDa and the overall predicted structure shows an N‐terminal death domain, two immunoglobulin domains, a paracaspase domain and another immunoglobulin‐like domain. The paracaspase domain is folded in a similar way to that of caspases and exhibits the ability to bind on substrate. Also, the enzyme, contrary to caspases, seems not to require cleavage of loop 4 to become active [37, 38].

The other member of CD clan of the cysteine proteases are the metacaspases (**Figure 1**). They are found in Fungi, Protozoa, Chromista and Plantae [8], and were first described in 2000 [32], on a study performing structural and sequence analysis, which revealed a great diversity of protease genes related to caspases in these Phyla. They were credited as strong candidates to perform central roles on PCD [11].

Based on their structural architecture, they were divided into two categories: type I and type II metacaspases. The type I subclass metacaspases have an N‐terminal extension, a prodomain with a proline‐rich repeat motif that is absent on the type II metacaspases, which, instead, present a 200 amino acid C‐terminal extension. Also, many plant type I metacaspases have a zinc finger motif that is similar to that of the plant hypersensitive response/cell death protein lsd‐1 [39].

Recently, a third group, named as type III metacaspases, has been proposed [40]. These proteins are found on many phytoplankton organisms and are not grouped with the other metacaspase groups, probably by a p10 domain rearrangement on which the motif SGCXDXQTSADV is located on the N‐terminus rather than on the C‐terminus, as usual for plant metacaspases. The studied organisms also revealed the presence of metacaspase‐like proteases which possess only the p10 domain. These proteases are found on bacteria and may represent an evolutionary connection.
