Marc Germain

Cleavage of Automodified Poly(ADP-ribose) Polymerase during Apoptosis EVIDENCE FOR INVOLVEMENT OF CASPASE-7

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADP-ribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.

Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis

The endoplasmic reticulum (ER) can elicit proapoptotic signalling that results in transmission of Ca2+ to the mitochondria, which in turn stimulates recruitment of the fission enzyme DRP1 to the surface of the organelle. Here, we show that BH3‐only BIK activates this pathway at the ER in intact cells, resulting in mitochondrial fragmentation but little release of cytochrome c to the cytosol. The BIK‐induced transformations in mitochondria are dynamic in nature and involve DRP1‐dependent remodelling and opening of cristae, where the major stores of cytochrome c reside. This novel function for DRP1 is distinct from its recognized role in regulating mitochondrial fission. Selective permeabilization of the outer membrane with digitonin confirmed that BIK stimulation results in mobilization of intramitochondrial cytochrome c. Of note, BIK can cooperate with a weak BH3‐only protein that targets mitochondria, such as NOXA, to activate BAX by a mechanism that is independent of DRP1 enzyme activity. When expressed together, BIK and NOXA cause rapid release of mobilized cytochrome c and activation of caspases.

BH3-only BIK Regulates BAX,BAK-dependent Release of Ca2+ from Endoplasmic Reticulum Stores and Mitochondrial Apoptosis during Stress-induced Cell Death

BIK, a pro-apoptotic BH3-only member of the BCL-2 family, targets the membrane of the endoplasmic reticulum (ER). It is induced in human cells in response to several stress stimuli, including genotoxic stress (radiation, doxorubicin) and overexpression of E1A or p53 but not by ER stress pathways resulting from protein malfolding. BIK initiates an early release of Ca2+ from ER upstream of the activation of effector caspases. Release of the mobile ER Ca2+ stores in baby mouse kidney cells doubly deficient in BAX and BAK, on the other hand, is resistant to BIK but is sensitive to ectopic BAK. Over-expression of p53 stimulates recruitment of BAK to the ER, and both its recruitment and assembly into higher order structures is inhibited by BIK small interfering RNA. Employing small interfering RNA knockdowns, we also demonstrated that release of ER Ca2+ and mitochondrial apoptosis in human epithelial cells requires BIK and that a Ca2+-regulated target, the dynamin-related GTPase DRP1, is involved in p53-induced mitochondrial fission and release of cytochrome c to the cytosol. Endogenous cellular BIK, therefore, regulates a BAX,BAK-dependent ER pathway that contributes to mitochondrial apoptosis.

Antagonism of Beclin 1‐dependent autophagy by BCL‐2 at the endoplasmic reticulum requires NAF‐1

In addition to mitochondria, BCL‐2 is located at the endoplasmic reticulum (ER) where it is a constituent of several distinct complexes. Here, we identify the BCL‐2‐interacting protein at the ER, nutrient‐deprivation autophagy factor‐1 (NAF‐1)—a bitopic integral membrane protein whose defective expression underlies the aetiology of the neurodegenerative disorder Wolfram syndrome 2 (WFS2). NAF‐1 contains a two iron–two sulphur coordinating domain within its cytosolic region, which is necessary, but not sufficient for interaction with BCL‐2. NAF‐1 is displaced from BCL‐2 by the ER‐restricted BH3‐only protein BIK and contributes to regulation of BIK‐initiated autophagy, but not BIK‐dependent activation of caspases. Similar to BCL‐2, NAF‐1 is found in association with the inositol 1,4,5‐triphosphate receptor and is required for BCL‐2‐mediated depression of ER Ca2+ stores. During nutrient deprivation as a physiological stimulus of autophagy, BCL‐2 is known to function through inhibition of the autophagy effector and tumour suppressor Beclin 1. NAF‐1 is required in this pathway for BCL‐2 at the ER to functionally antagonize Beclin 1‐dependent autophagy. Thus, NAF‐1 is a BCL‐2‐associated co‐factor that targets BCL‐2 for antagonism of the autophagy pathway at the ER.

Reactive Oxygen Species: Stuck in the Middle of Neurodegeneration

Neuronal cell loss associated with neurodegeneration has recently been linked to mitochondrial dysfunction. Electron transport chain defects and reactive oxygen species (ROS) production are emerging as important players in the etiology of neurodegenerative diseases. Proper management of ROS and disposal of damaged cellular components are vital to the survival and function of neurons. Proteins involved in these pathways are often mutated in neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, amyotrophic lateral sclerosis, and Huntingtons disease. In this review, we will discuss the roles of ROS in normal physiology, how changes in ROS production affect neuronal survival in neurodegenerative diseases, and the recent advances in mitochondrial antioxidants as potential therapeutics.

Induction and endoplasmic reticulum location of BIK/NBK in response to apoptotic signaling by E1A and p53

A DNA microarray analysis identified the BH3-only BCL-2 family member, BIK/NBK, as a transcript that is upregulated during induction of apoptosis by oncogenic E1A. E1A depended on wild-type p53 to induce BIK and activate the death program. Further, p53 independently induced BIK RNA and protein, and BIK alone stimulated cell death in p53-null cells, dependent on the activation of caspases. BIK function, however, was abrogated by a disabling point mutation within the BH3 domain. Collectively, these results argue that BIK is a downstream apoptotic effector of p53 in response to a physiological p53-mediated death stimulus provided by E1A. Elevated BCL-2 functioned downstream of p53 and BIK induction to inhibit the E1A death pathway, with the ratio of anti-apoptotic BCL-2 and pro-apoptotic BIK determining cell death or survival in E1A-expressing cells. Cells expressing BCL-2 or treated with the pan caspase inhibitor, zVAD-fmk, allowed accumulation of high levels of cytotoxic BIK compared to control cells. Of note, a significant fraction of either ectopic or endogenous BIK was found associated with the endoplasmic reticulum, suggesting that this organelle, in addition to mitochondria, may be a target of BIK function.

MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL‐2 homologue MCL‐1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL‐1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL‐1 levels regulate activation of autophagy. Furthermore, deletion of MCL‐1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin‐1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL‐1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1s role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation‐induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein‐dependent manner.

BH3-ligand regulates access of MCL-1 to its E3 ligase

A genome wide search for new BH3‐containing Bcl‐2 family members was conducted using position weight matrices (PWM) and identified a large (480 kDa), novel BH3‐only protein, originally called LASU1 (now also known as Ureb‐1, E3histone, ARF‐BP1, and Mule). We demonstrated that LASU1 is an E3 ligase that ubiquitinated Mcl‐1 in vitro and was required for its proteasome‐dependent degradation in HeLa cells. Of note, the BH3 domain of LASU1 interacted with Mcl‐1 but not with Bcl‐2 or Bcl‐Xl. A competing BH3‐ligand derived from Bim interacted with Mcl‐1 and prevented its interaction with LASU1 in HeLa cells, causing elevation of the steady‐state levels of Mcl‐1. This suggests that the unliganded form of Mcl‐1 is sensitive to LASU1‐mediated degradation of Mcl‐1.

Proteolysis of poly(ADP-ribose) polymerase by caspase 3 : Kinetics of cleavage of mono(ADP-ribosyl)ated and dna-bound substrates

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme which is responsible for synthesis of poly(ADP-ribose) in response to DNA damage caused by numerous agents and during DNA base excision repair. After DNA damage, the enzyme binds to nicks in DNA through its N-terminal zinc fingers and catalyzes the formation of poly(ADP-ribose) on various nuclear acceptors including itself. When DNA damage is extensive, cells induce their own demise by activating the proteases that induce apoptosis (caspases) which cleave PARP and other death substrates. Here we report the development of a new approach to investigate the sensitivity of mono(ADP-ribosyl)ated and DNA-bound PARP to cleavage during apoptosis. The development of a stoichiometric labeling procedure of the enzyme has allowed us to evaluate the catalytic properties of caspase 3 toward mono(ADP-ribosyl)ated PARP at various enzyme:substrate molar ratios. We show that low levels of automodification (< or = 3 U of ADP-ribose per chain) do not inhibit the proteolysis of the substrate. In addition, we demonstrate that binding of unmodified PARP to DNA influences the kinetics of its cleavage by caspase 3.