Summary of Classic Signal Pathways - Caspase Protein Family

Caspase is a group of proteases existing in the cytoplasm. Its full name is cysteinyl aspartate specific proteinase. One of the important common features of caspases is that their active sites all contain cysteine, which enables them to specifically cleave peptide bonds on the aspartic acid residues of target proteins. This allows caspases to highly selectively cleave certain proteins. They play a significant role in programmed cell death (including apoptosis, pyroptosis, and necroptosis, etc.) and inflammation. Currently, 11 different caspases have been identified in humans. The mammalian Caspase family proteins can be classified into three major categories based on their structure and function.

The first category: pro-apoptotic Caspases

This category mainly includes Caspase-2, 8, 9, 10, 3, 6, and 7. They efficiently cleave intracellular proteins in a cascade amplification manner, ultimately leading to apoptosis. They are divided into two groups. Among them, Caspase 2, 8, 9, and 10 are involved in the initiation of apoptosis (initiator) and can self-activate in the presence of pro-apoptotic signals and activate downstream Caspases; Caspase 3, 6, and 7 are involved in the execution of apoptosis and can be activated by the upstream initiator, and after activation, they act on specific substrates to cause biochemical and morphological changes in the cell, leading to apoptosis.

Type II: Pro-inflammatory Caspases

These mainly include Caspase 1, 4, 5, 11, 12, and 13. During the activation of innate immune responses, pro-inflammatory Caspases mediate the maturation of specific cytokines, such as Pro-IL-1β and Pro-IL-18, to form active IL-1β and IL-18; they may also be involved in a form of cell death called pyroptosis.

The third category: Keratinocyte differentiation

The main actor is Caspase-14. Caspase-14 is a cysteine protease that seems non-apoptotic. Its main function is to be expressed in the basal epidermal layer and be activated during the keratinization process of keratinocytes, participating in the differentiation and keratinization of keratinocytes.

Functional classification of Caspase in mammals

Shi Y. Mechanisms of Caspase Activation and Inhibition during Apoptosis. Molecular Cell, Vol. 9, 459–470, March, 2002.

1. Caspases are mediators of apoptosis and inflammation, they have become attractive targets for the treatment of various diseases including neurodegenerative disorders, inflammation, metabolic diseases and cancer:

①    Caspase-1

A protease that plays an important role in the process of apoptosis and inflammation. Its main functions include participating in the activation of pro-inflammatory cytokines, apoptosis, and pyroptosis. Caspase-1 is directly or indirectly related to the occurrence of diseases such as neurological disorders, cardiovascular diseases, metabolic diseases, ophthalmic diseases, and chronic liver failure. Currently, small molecule drugs CTS-2090, CTS-2096, ML-132, TMC-Ag5 targeting Caspase-1 are in preclinical research, and Q-004 is in the stage of drug discovery.

②    Caspase-3

The key proteases for apoptosis in mammalian cells, by specifically cleaving key proteins, lead to DNA lysis and thereby promote cell apoptosis. This enzyme is closely related to the occurrence and development of various diseases, including neurodegenerative diseases, various cancers such as breast cancer and ovarian cancer, cardiovascular diseases and metabolic diseases. It is one of the most popular and extensively studied targets. SM-1, Suftalan Zinc, and NWL-283 are all Caspase-3 inhibitors, among which SM-1 is in the third phase of clinical research.

③    Caspase-8

It plays a key role in cell apoptosis and immune regulation, and is the initiator of the extrinsic apoptotic pathway. It not only induces cell apoptosis but also participates in regulating the transformation between different cell death patterns. Studies have found that the abnormality of Caspase-8 is closely related to various diseases, including cancers such as breast cancer, lung cancer, and liver cancer, neurodegenerative diseases, and immune system disorders. Currently, according to the information from the New Drug Repository, there are no small molecule drugs targeting Caspase-8 under development.

④    Caspase-9

As a key regulatory factor of apoptosis, it plays an important role in maintaining cellular homeostasis and preventing abnormal proliferation. When cells are subjected to internal stress or damage, Caspase-9 is activated, triggering the apoptotic program and eliminating damaged cells. Studies have shown that Caspase-9 is closely related to the occurrence and development of various diseases such as neurodegenerative diseases, cancer, and immune system diseases. Currently, there is a small molecule drug OT-717 in preclinical research.

2. Caspase substrate

It generally consists of two parts: Caspase recognition sequence and signal generation (chromogenic substrate and fluorescent group). Different Caspase recognition sequences are different. Caspase enzyme recognition sequences are generally composed of three or four amino acids, and the N-terminus of the enzyme recognition sequence is usually modified with acetyl (Ac) or carboxymethoxy (Z) groups to increase membrane permeability. The corresponding Caspase recognizes specific peptide sequences as its enzymatic cleavage sites, releasing motifs that generate or inhibit signals. The chromogenic and fluorescent substrates of Caspase function in a similar manner, where the signal or color intensity of the substrate is directly proportional to its proteolytic activity.

Caspase chromogenic substrate: they are composed of Caspase recognition sequences and chromophores. Common chromophores include pNA para nitroaniline or 4-nitroaniline, which can be detected by enzyme-linked immunosorbent assay (ELISA) or spectrophotometer at 405 nm for optical density.

Fluorescent substrates of Caspase: Their structure contains fluorescent groups related to recognition by caspase, such as 7-amino-4-methylcoumarin (AMC), 7-amino-4-trifluoromethylcoumarin (AFC), and Caspase substrates labeled with AMC and AFC are commonly used for kinetic assays.

Caspase Inhibitors: They are composed of Caspase recognition sequence and protease inhibition motif. Caspase inhibitors can bind to the active site of Caspase and form reversible or irreversible connections. The C-terminus of Caspase recognition sequence is usually composed of functional groups such as aldehyde (- CHO) or fluoromethylketone (- FMK). Cystatine inhibitors with aldehyde functional groups are reversible, while inhibitors with FMK are irreversible. Both substrates and inhibitors of caspase have minimal cytotoxic effects, making them useful tools for studying caspase activity.