Article

Humanin Binds and Nullifies Bid Activity by Blocking Its Activation of Bax and Bak

Burnham Institute, La Jolla, California 92037, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(16):15815-24. DOI: 10.1074/jbc.M411902200
Source: PubMed

ABSTRACT

Recently, we discovered that Humanin (HN), a small endogenous peptide of 24 amino acids, binds to and inhibits the proapoptotic
protein Bax. We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form
of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein. Synthetic HN peptide binds purified
Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria, whereas mutant peptides that fail to bind Bid or tBid lack this activity. Moreover,
HN peptide also retained protective activity on bax–/–mitochondria, indicating that HN can block tBid-induced release of apoptogenic proteins from these organelles in a Bax-independent
manner. HN peptide inhibits tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, as shown by experiments
with chemical cross-linkers or gel filtration. Gene transfection experiments showed that HN (but not an inactive mutant of
HN) also protects intact cells from apoptosis induced by overexpression of tBid. We conclude that Bid represents an additional
cellular target of HN, and we propose that HN-mediated suppression of Bid contributes to the antiapoptotic activity of this
endogenous peptide.

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    • "This suggests that the interaction between rat HN and BAX may prevent targeting of BAX to the mitochondria to initiate cell death. In this study, we have validated and expanded a previous observation that HN binds BAX in vitro (Luciano et al., 2005; Zhai et al., 2005) to prevent apoptosis of neuronal cells in culture. We have shown for the first time that when BAX was associated with rat HN, HN : BAX complex is not translocated to the mitochondria in vivo in the testis. "
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    ABSTRACT: We have previously demonstrated that the mitochondria-derived cytoprotective peptide humanin (HN), when administered intratesticularly to rats, rescues germ cells from apoptosis secondary to testicular stress of hormonal deprivation induced by gonadotropin-releasing hormone antagonist (GnRH-A). To decipher the cellular mechanisms of HN action in the amelioration of GnRH-A-induced germ cell apoptosis, adult male rats received the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of GnRH-A on Day 1 and daily IT injection of saline; (iii) daily IT injection of synthetic HN; and (iv) GnRH-A injection on Day 1 and daily IT injection of HN (GnRH-A+HN). HN alone had no effect on germ cell apoptosis. GnRH-A increased germ cell apoptosis and BAX in the testicular mitochondrial fractions. Synthetic HN decreased germ cell apoptosis induced by GnRH-A and BAX in the mitochondria. We deduced that the cytoprotective action of synthetic HN on GnRH-A-induced germ cell apoptosis was mediated by attenuating p38 mitogen-activated protein kinase activity and increasing STAT3 phosphorylation. The effect of synthetic HN on the expression of endogenous rat HN in the testis was studied using rat HN specific antibody. GnRH-A treatment increased, but concomitant treatment with synthetic HN reduced endogenous rat HN expression in both cytosolic and mitochondrial fractions in testis. Co-immunoprecipitation experiments demonstrated that the increased rat HN was physically associated with BAX in the cytosolic testicular fractions after GnRH-A treatment. Double-immunofluorescence staining confirmed the co-localization of BAX and rat HN in the cytoplasm of Leydig cells and spermatocytes after GnRH-A treatment. We conclude that the cytoprotective effect of exogenously administered synthetic HN is mediated by interactions of endogenous rat HN with BAX in the cytoplasm preventing the entry of BAX to the mitochondria to govern the fate of germ cell survival or death during pro-apoptotic stress to the testis in rats.
    Full-text · Article · May 2013 · Andrology
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    • "Recently, alternative mechanisms of action for transfected HN showed that it binds to Bax, BimEL, or tBid, and prevents translocation of these proteins from cytoplasm to mitochondria, thereby protecting cells from apoptosis-inducing insults that trigger the mitochondrial pathway for cell death [18] [19] [20]. However, the therapeutic application of HN in the treatment of neurodegenerative diseases is limited due to its rapid degradation in vivo and poor ability to cross the blood–brain barrier (BBB) and enter the cytoplasm of neuronal cells where cell death occurs. "
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    ABSTRACT: Inhibition of the early intracellular event that triggers neurodegenerative cascades and reversal of neuronal cell death are essential for effective treatment of Alzheimer's disease (AD). In this study, a novel therapeutic for AD, a transducible humanin with an extended caspase-3 cleavage sequence (tHN-C3), was developed and showed multiple mechanisms of therapeutic action. These included targeted delivery of anti-apoptotic protein humanin through the blood-brain barrier (BBB) to neuronal cells, specific inhibition of caspase-3 activation to inhibit the early triggering of AD progression, and delivery of humanin into the cytoplasm of neuronal cells undergoing apoptosis where it exerts its anti-apoptotic functions effectively. The tHN-C3 prevented neuronal cell death induced by H(2)O(2), or soluble Aβ(42), via Bax binding. In animal models of AD induced by amyloid beta, in Tg2576 mice, and in the rat middle cerebral artery occlusion model of stroke, tHN-C3 effectively prevented neuronal cell death, inflammatory cell infiltration into the brain, and improved cognitive memory. The therapeutic effectiveness of tHN-C3 was comparable to that of Aricept, a clinically approved drug for AD treatment. Therefore, tHN-C3 may be a new remedy with multiple therapeutic functions targeting the early and late stages of neurodegeneration in AD and other brain injuries.
    Full-text · Article · Jan 2013 · Journal of Controlled Release
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    • "Recently, alternative mechanisms of action for transfected HN showed that it binds to Bax, BimEL, or tBid, and prevents translocation of these proteins from cytoplasm to mitochondria, thereby protecting cells from apoptosis-inducing insults that trigger the mitochondrial pathway for cell death [18] [19] [20]. However, the therapeutic application of HN in the treatment of neurodegenerative diseases is limited due to its rapid degradation in vivo and poor ability to cross the blood–brain barrier (BBB) and enter the cytoplasm of neuronal cells where cell death occurs. "

    Full-text · Article · Jan 2013 · Journal of Controlled Release
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