Key Targets in Apoptosis, Necroptosis, and Autophagy: Novus Biologicals

Cell death/recycling pathways such as apoptosis, necroptosis, and autophagy are an integral part of the growth, development, homeostasis as well as the pathophysiology in the life of living organisms. These signaling pathways are highly regulated and some of their key regulatory targets are discussed below.

Apoptosis

Apoptosis, programmed cell death, is primarily characterized by the activation of caspases which further regulate the mass cleavage of proteins and DNA. Some of major the proteins responsible for various apoptotic events are:

Initiator Caspases (-2, -8, -9, -10)	In apoptosis, initiator caspases are involved in the upstream events of death receptor (extrinsic)- or mitochondrion-dependent (intrinsic) signaling pathways. They are responsible for the cleavage of executioner caspases.
Executioner Caspases (-3, -6, -7)	Upon activation by initiator caspases, executioner or effector caspases orchestrate the cell death process by cleaving many different cellular proteins. They cleave PARP and inhibit its DNA-nicks repairing potential.
Death receptors & ligands	In the extrinsic pathway, the death ligands TRAIL, FASL, and TNF-alpha bind to the receptors TRAILR1/2 (DR4/DR5), FAS/CD95 and TNFRI respectively and initiate the activation of Caspase-8 and -10.
Bcl-2 Proteins	Intrinsic pathway signals activate the pro-apoptotic Bcl-2 proteins (Bax, Bak, BID, BAD, PUMA, NOXA or BIM) and neutralize the antiapoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, MCL1) leading to Cytochrome-c/Smac release via disruption of mitochondrial integrity. This is followed by apoptosome-dependent Caspase -9 activation.

Necroptosis

Necroptosis, programmed necrosis, is a caspase-independent form of cell death which is driven by the activation of receptor-interacting serine/threonine-protein kinases (RIPs) and mixed lineage kinase domain-like protein (MLKL). Some of the key proteins which are critical in necroptosis are:

Receptors	Upon binding to their ligands, receptors such as the TNF-alpha receptor superfamily, T-cell receptors (TCRs), interferon receptors (IFNRs) and Toll-like receptors (TLRs) drive necroptosis signaling.
Necrosome	RIP1, RIP3 and MLKL generates a complex which is called the necrosome or RIP1-RIP3-MLKL complex. The necrosome regulates upstream cell death receptors and downstream executional targets, as well as events including ROS burst, plasma membrane permeabilization, and cytosolic ATP reduction.
RIP1-RIP3-MLKL phosphorylations	RIP1 activation during necroptosis triggers RIP3 phosphorylation and subsequent phosphorylation of MLKL. Trimerized phospho-MLKL translocates to the plasma membrane inducing necrotic plasma membrane permeabilization. Some inducers may bypass RIP1 for direct activation of RIP3 or MLKL.

Caspase 3 antibody

MLKL antibody

Caspase 3 protein was analyzed in control and 2mM Staurosporine-treated HeLa cells. Immunoblotting was performed using Caspase-3 antibody (clone 31A1067) [NB100-56708] and the signal was developed with the ECL-based method. This antibody recognized both the pro- and the cleaved forms of Caspase 3 in samples from 2-12 hour treatment time points which signifies apoptosis induction.

Phospho-MLKL (pMLKL) was detected in formalin fixed paraffin-embedded sections of human squamous cell carcinoma using pThr357-MLKL antibody (clone 954724) [MAB91871] at 5 ug/ml concentration. The signal was developed with anti-mouse IgG VisUCyte HRP Polymer antibody and DAB (brown), which followed counterstaining with hematoxylin (blue). pMLKL signal was very intense in the membranes of apparently necroptic cells.

Autophagy

Autophagy, autophagocytosis, is a cell’s mechanism of regulated destruction of its proteins and other components including organelles. It is characterized by the lipidation of LC3-I to LC3-II and then LC3-II’s translocation (along-with p62) to the membranes of autophagosomes. Some of the key proteins which drive autophagy events are:

LC3	LC3, a ubiquitin-like modifier, is critical to the formation of autophagosomes. During autophagy, LC3B-I (cytosolic) is lipidated to form LC3B-II which incorporates into the autophagosomal membranes. Conversion of LC3-I to LC3-II is a gold standard marker of autophagy induction.
P62/SQSTM1	p62 is a stress-inducible protein with multiple domains/binding partners. In addition to its role as a signaling hub for amino acid sensing and the oxidative stress response, p62 acts as a selective autophagy receptor for degradation of ubiquitinated substrates.
ATG5	During autophagosome biogenesis, ATG5 binds ATG12. Thereafter, the ATG5-ATG12 conjugate binds to ATG16L1 to generate the autophagy elongation complex (ATG5-12/16L1) which regulates lipidation of LC3.
Beclin 1	It is a mammalian ortholog of yeast Atg6 protein. Beclin 1 positively regulates autophagy through activation of Vps34.