Novel Death Defying Domain in met entraps the active site of caspase-3 and blocks apoptosis in hepatocytes

Abstract

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF) is known to function as a potent anti-apoptotic mediator in normal and neoplastic cells. Herein we report that intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of Caspase-3 cleavage sites, which bait, trap and disable the active site of Caspase-3, thereby blocking the execution of apoptosis. We call this Caspase-3 cleavage motif the ‘Death Defying Domain’ (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by Caspase-3, the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of Caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of Caspase-3 activity. By gain and loss-of-function studies using restoration of DDD expression in DDD deficient hepatocytic cells, we found that both Caspase-3 sites in DDD are necessary for inhibition of Caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met’s enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of Caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion Our findings show that Met can directly inhibit Caspase-3 via a novel mechanism and promote hepato-cyte survival. Results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play.

The differential effects of high-fat and high fructose diets on the liver of male albino rat and the proposed underlying mechanisms

“…Caspase is a unique set of cysteine proteases that cleave critical cellular proteins [35]. Caspase 3 is the key killer of apoptosis, thus promoting cell survival [23]. The value of the caspase 3 increased markedly in the HFD, HFruD and HFHF groups.…”

Genomic Instability Causes HGF Gene Activation in Colon Cancer Cells, Promoting Their Resistance to Necroptosis

“…HGF exerts its biological effects via its tyrosine kinase cell surface receptor known as Met 1–4 . When aberrantly activated, however, the HGF-Met pathway could act as an oncogenic “driver” and promote tumorigenesis 1, 2 .…”

How does met regulate the survival/apoptosis balance?

“…2 However, recent findings demonstrated that Met can regulate survival/apoptosis balance through an unexpected mechanism. Indeed, the team of R. Zarnegar 3 showed in an article in HEPATOLOGY that the human Met receptor displayed at its C-terminal tail a double consensual caspase site able to inhibit caspase 3 activity, a mechanism reminiscent of Nevertheless, we previously demonstrated that during apoptosis Met is cleaved by caspases at another site located in the juxtamembrane region. 4 This cleavage generates a 40-kDa fragment found in apoptotic murine hepatocytes in vivo.…”

“…3 Thus, the overexpression of Met often observed in HCC could directly inhibit caspase activity, contributing to apoptosis resistance. It would be now interesting to evaluate whether, in the opposite manner, the proapoptotic juxtamembrane cleavage of Met is inhibited in HCC.…”

“…To confirm this hypothesis, the estimated glomerular filtration rate (eGFR), 3 hemoglobin (Hb), and RBV plasma concentration in 23 HCV genotype 1-infected patients treated with fixed doses of the triple therapy for 12 weeks and, successively, with the dual therapy until 48 weeks, were evaluated at weeks 4, 8, 12, 24, and 48.…”