Protein myristoylation in health and disease

N-myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal glycine residue of target proteins, catalysed by N-myristoyltransferase (NMT), a ubiquitous and essential enzyme in eukaryotes. Many of the target proteins of NMT are crucial components of signalling pathways, and myristoylation typically promotes membrane binding that is essential for proper protein localisation or biological function. NMT is a validated therapeutic target in opportunistic infections of humans by fungi or parasitic protozoa. Additionally, NMT is implicated in carcinogenesis, particularly colon cancer, where there is evidence for its upregulation in the early stages of tumour formation. However, the study of myristoylation in all organisms has until recently been hindered by a lack of techniques for detection and identification of myristoylated proteins. Here we introduce the chemistry and biology of N-myristoylation and NMT, and discuss new developments in chemical proteomic technologies that are meeting the challenge of studying this important co-translational modification in living systems.     

Caspase knockouts: matters of life and death

Apoptosis, the seemingly counter-intuitive act of physiological cell suicide, is accomplished by an evolutionarily conserved death program that is centered on the activation of a group of intracellular cysteine proteases known as caspases. It is now clear that both extra- and intra-cellular stimuli induce apoptosis by triggering the activation of these otherwise latent proteases in a process that culminates in caspase-mediated disintegration of cellular contents and their subsequent absorption by neighboring cells. While many elegant in vitro studies have demonstrated the requirement of caspase activities for the execution of most, if not all, apoptosis, the precise contribution of individual caspases in vivo and how they functionally relate to each other remain poorly elucidated. Fortunately, the generation of various caspase deficient mice through gene targeting has provided a unique window of opportunity to definitely examine the physiological function of these caspases in vivo. As the list of caspase knockouts grows, we considered it was time to review what we have been learned, from these studies about the exact role of individual caspases in mediating apoptotic events. We will also provide our prediction on the direction of future studies in this ever-growing field of caspases.     

Origins of signalling and memory: matters of life versus death

This review focuses on the principles in cell-cell communication and cellular ability to respond to external chemical changes which have been so crucial for the development of life on planet Earth. We now know that the capacity of free-living organisms which evolved more than a billion years ago to respond to intercellular signal molecules, originating either from themselves or from other sources in their vicinity, is so similar possibly even more sophisticated - to that of the cells in our own body, and these findings have had a major impact on our struggle to understand how life has evolved and how it can be maintained. Attention is drawn to the very important topic of mechanisms in cell death, being seen as an aggressive and very powerful instrument in the continuance of life and ability of life to proliferate into a plethora of new species, and use insulin-related material as our paradigm. Such signal molecules (hormones) may have played a major role in cellular maintenance throughout evolution.     

Yeast endonuclease G: complex matters of death, and of life

In a recent issue of Molecular Cell, Büttner et al. (2007) described dual roles for the conserved mitochondrial protein endonuclease G in budding yeast apoptosis and proliferation that illuminate some of the contradictory and confusing findings regarding this protein in mammalian cells.     

Mechanisms of cell death of thymocytes induced by polyunsaturated, monounsaturated and trans-fatty acids

Polyunsaturated fatty acids (PUFAs) are rapidly cytotoxic to isolated murine thymocytes, and the degree of cell death has been correlated with changes in membrane fluidity, elevation of intracellular calcium concentration and generation of reactive oxygen species. We have compared the degree of cell death and increase in membrane fluidity of C-20 and C-22 omega-3 and 6 PUFAs to those induced by monounsaturated and trans-fatty acids, and find that concentrations which induce comparable increases in membrane fluidity do not cause comparable cell death. The C-18 omega-6 causes a decrease in membrane fluidity, yet is the most potent in causing cell death. Omega-6 PUFAs are more cytotoxic than omega-3 PUFAs, while monounsaturated and trans-fats show little cytotoxicity and only at much higher concentrations. Cell death is preceded by reductions of both plasma and mitochondrial membrane potential, and occurs via apoptosis. These results indicate that cell death is due to mechanisms other than changes in membrane fluidity.     

The myristoylation site of meiotic lamin C2 promotes local nuclear membrane growth and the formation of intranuclear membranes in somatic cultured cells

Lamin C2 is a splice product of the mammalian lamin A gene and expressed in primary spermatocytes where it is distributed in the form of discontinuous plaques at the nuclear envelope. We have previously shown that the aminoterminal hexapetide GNAEGR of lamin C2 following the start methionine is essential for its association with the nuclear envelope and that the aminoterminal glycine of the hexapeptide is myristoylated. Here we have analyzed the ultrastructural changes induced in COS-7 and Xenopus A6 cells by overexpressing rat lamin C2 or a human lamin C mutant possessing the lamin C2-specific hexapeptide at its aminoterminus. Both lamins were targeted to the nuclear envelope of mammalian and amphibian cells and induced the formation of intranuclear membranes, whereas wild-type human lamin C and a lamin C2 mutant, that both lack this lipid moiety, did not. Our data indicate that the myristoyl group of lamin C2 has besides its demonstrated role in nuclear envelope association additional functions during spermatogenesis. Our present study complements previously published results where we have shown that the CxxM motif of lamins promotes nuclear membrane growth (Prüfert et al., 2004. J. Cell Sci. 117, 6105-6116).     

Single-step purification of myristoylated and nonmyristoylated recoverin and substrate dependence of myristoylation level

Recoverin is cotranslationally modified by the covalent linkage of a myristoyl group to its N terminus. It is a member of a family of Ca(2+)-myristoyl switch proteins. Recombinant myristoylated revoverin is currently produced by the cotransformation of bacteria with recoverin and an enzyme that allows N-myristoylation and by supplementing the culture medium with myristic acid. A large variation in the myristoylation level of recoverin and in the amount of myristic acid supplied to the culture medium can be found in the literature. Moreover, although it is known to strongly affect bacterial growth, the amount of ethanol used to solubilize myristic acid is only scarcely mentioned. To improve our understanding of the parameters responsible for recombinant recoverin myristoylation, the effects of myristic acid and ethanol on recoverin myristoylation and expression levels have been systematically studied. In addition, a single-step purification procedure to produce purified myristoylated and nonmyristoylated recombinant recoverin has also been devised. Finally, sodium myristate has been used as an efficient alternative substrate to achieve high myristoylation and expression levels of recoverin. Given that a large number of proteins are myristoylated, these procedures could be applied to several other proteins in addition to recoverin.     

Mitochondrial role in life and death of the cell

Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.     

Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction

Cells and tissues are exposed to multiple mechanical stresses during development, tissue homoeostasis and diseases. While we start to have an extensive understanding of the influence of mechanics on cell differentiation and proliferation, how excessive mechanical stresses can also lead to cell death and may be associated with pathologies has been much less explored so far. Recently, the development of new perturbative approaches allowing modulation of pressure and deformation of tissues has demonstrated that compaction (the reduction of tissue size or volume) can lead to cell elimination. Here, we discuss the relevant type of stress and the parameters that could be causal to cell death from single cell to multicellular systems. We then compare the pathways and mechanisms that have been proposed to influence cell survival upon compaction. We eventually describe the relevance of compaction‐induced death in vivo, and its functions in morphogenesis, tissue size regulation, tissue homoeostasis and cancer progression.     

Estradiol rescues neurons from global ischemia-induced cell death: Multiple cellular pathways of neuroprotection

The potential neuroprotective role of sex hormones in chronic neurodegenerative disorders and acute brain ischemia following cardiac arrest and stroke is of a great therapeutic interest. Long-term pretreatment with estradiol and other estrogens affords robust neuroprotection in male and female rodents subjected to focal and global ischemia. However, the receptors (e.g., cell surface or nuclear), intracellular signaling pathways and networks of estrogen-regulated genes that intervene in neuronal apoptosis are as yet unclear. We have shown that estradiol administered at physiological levels for two weeks before ischemia rescues neurons destined to die in the hippocampal CA1 and ameliorates ischemia-induced cognitive deficits in ovariectomized female rats. This regimen of estradiol treatment involves classical intracellular estrogen receptors, transactivation of IGF-1 receptors and stimulation of the ERK/MAPK signaling pathway, which in turn maintains CREB activity in the ischemic CA1. We also find that a single, acute injection of estradiol administrated into the brain ventricle immediately after an ischemic event reduces both neuronal death and cognitive deficits. Because these findings suggest that hormones could be used to treat patients when given after brain ischemia, it is critical to determine whether the same or different pathways mediate this form of neuroprotection. We find that an agonist of the membrane estrogen receptor GPR30 mimics short latency estradiol facilitation of synaptic transmission in the hippocampus. Therefore, we are testing the hypothesis that GPR30 may act together with intracellular estrogen receptors to activate cell signaling pathways to promote neuron survival after global ischemia.     

Heat shock proteins,cellular chaperones that modulate mitochondrial cell death pathways

Stress or heat shock proteins (HSPs) are ubiquitous and highly conserved proteins whose expression is induced in response to a wide variety of physiological and environmental insults. They allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. These proteins play an essential role in intracellular "house-keeping" by assisting the correct folding of nascent and stress-accumulated misfolded proteins and preventing their aggregation. Several HSPs have also demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream, and downstream of the mitochondrial events. Finally, HSPs could play a role in the proteasome-mediated degradation of selected proteins under stress conditions. Altogether, these properties could make HSPs appropriate targets for modulating cell death pathways.     

Sheathing the swords of death: Post-translational modulation of plant metacaspases

Plant metacaspases (MCPs) are conserved cysteine proteases that have been postulated as regulators of programmed cell death (PCD). Although MCPs have been proven to have PCD relevant functions in multiple species ranging from fungi to plants, how these proteases are modulated in vivo remains unclear. Aside from demonstrating that these proteases are distinct from metazoan caspases due to their different target site specificities, how these proteases are used to tightly regulate cell death progression is a key question that remains to be resolved. Some recent studies on the biochemical characteristics of type-II MCP activities in Arabidopsis may begin to shed additional light on this aspect. The in vitro catalytic activities of recombinant AtMC4, AtMC5 and AtMC8 are found to be Ca²⁺-dependent while recombinant AtMC9 is active under mildly acidic conditions and not dependent on stimulation by Ca²⁺. Alterations of cellular pH and Ca²⁺ concentration commonly occur during various stresses and may help to orchestrate differential activation of latent MCPs under these conditions. Recent peptide mapping for recombinant AtMC4 (also called Metacaspase-2d) followed by site-specific mutagenesis studies have revealed multiple potential self-cleavage sites with the identification of a conserved lysine residue (Lys-225) as the key position for enzyme function both in vitro and in vivo. The multiple self-cleavage sites in MCPs may also facilitate desensitization of these proteases and can provide a means for fine-tuning their proteolytic activities in order to achieve more sensitive control of downstream processes.     

Cheating death at the dawn of life: developmental control of apoptotic repression in the preimplantation embryo

During early development, the mammalian embryo is resistant to pro-apoptotic signals because of biochemical properties of the mitochondrion and nucleus. Mitochondria of the bovine two-cell embryo are resistant to depolarization because of low amounts of the proapoptotic protein BAX and high concentrations of the anti-apoptotic protein BCL2. As development proceeds, BAX content increases, BCL2 content declines, and mitochondria becomes capable of pore formation and depolarization in response to pro-apoptotic signals. The nucleus of the two-cell embryo is resistant to degradation by the DNase DFFB because epigenetic modifications, including DNA methylation and histone deacetylation, mask internucleosomal sites for DNA cleavage. Blastomere DNA becomes progressively less methylated during development so that DNA becomes accessible to cleavage by DFFB.     

Apoptosis: Programmed cell death in health and disease

Apoptosis is a normal physiological cell death process of eliminating unwanted cells from living organisms during embryonic and adult development. Apoptotic cells are characterised by fragmentation of nuclear DNA and formation of apoptotic bodies. Genetic analysis revealed the involvement of many death and survival genes in apoptosis which are regulated by extracellular factors. There are multiple inducers and inhibitors of apoptosis which interact with target cell specific surface receptors and transduce the signal by second messengers to programme cell death. The regulation of apoptosis is elusive, but defective regulation leads to aetiology of various ailments. Understanding the molecular mechanism of apoptosis including death genes, death signals, surface receptors and signal pathways will provide new insights in developing strategies to regulate the cell survival/death. The current knowledge on the molecular events of apoptotic cell death and their significance in health and disease is reviewed.     

Life and death decisions: the role of the IAPs in modulating programmed cell death

Multicellular organisms have evolved elaborate signal transduction pathways for maintaining homeostasis through the control of cell proliferation and death. The recent surge of interest in the regulation of programmed cell death has led to the rapid identification of many proteins involved in controlling and executing apoptosis. The inhibitors of apoptosis proteins (IAPs) constitute a family of highly conserved death suppressing proteins that were first identified in baculoviruses, and that has recently expanded to include at least two homologues in Drosophila melanogaster and four in rodents and humans. In this article we review the current state of IAP research. Two of the IAPs, HIAP-1 and HIAP-2, have been placed within the TNFα induced cell death pathway which involves two receptors for TNFα and multiple, overlapping signal transduction proteins. A third, X-linked gene termed XIAP, is ubiquitously expressed and appears to have a broad range of suppressor activity to a variety of apoptotic triggers. The fourth member, NAIP, has been identified as the protein product of a candidate gene for the inherited neuromuscular disorder, spinal muscular atrophy (SMA). The neuroprotective activity of NAIP in an in vivo model of cerebral ischemia has also been demonstrated. This revised version was published online in November 2006 with corrections to the Cover Date.     

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.     

Uncovering the role of VDAC in the regulation of cell life and death

Proper cell activity requires an efficient exchange of molecules between mitochondria and cytoplasm. Lying in the outer mitochondrial membrane, VDAC assumes a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. As such, it has been recognized that VDAC plays a crucial role in regulating the metabolic and energetic functions of mitochondria. Indeed, down-regulation of VDAC1 expression by shRNA leads to a decrease in energy production and cell growth. VDAC has also been recognized as a key protein in mitochondria-mediated apoptosis through its involvement in the release of apoptotic proteins located in the inter-membranal space and as the proposed target of pro- and anti-apoptotic members of the Bcl2-family and of hexokinase. Questions, however, remain as to if and how VDAC mediates the transfer of apoptotic proteins from the inter-membranal space to the cytosol. The diameter of the VDAC pore is only about 2.5–3 nm, insufficient for the passage of a folded protein like cytochrome c. New work, however, suggests that pore formation involves the assembly of homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins, such as Bax. Thus, VDAC appears to represent a convergence point for a variety of cell survival and cell death signals. This review provides insight into the central role of VDAC in mammalian cell life and death, emphasizing VDAC function in the regulation of mitochondria-mediated apoptosis and, as such, its potential as a rational target for new therapeutics.