The extended family of neutral sphingomyelinases

The neutral sphingomyelinases (N-SMases) are considered major candidates for mediating the stress-induced production of ceramide, and N-SMase activity has been identified, characterized, and cloned from bacteria, yeast, and mammalian cells. Although the level of identity between these enzymes is low, a number of key residues thought to be involved in metal binding and catalysis are conserved. This has led to the suggestion of a common catalytic mechanism, and thus, these enzymes are considered to form an extended family of N-SMases. Despite considerable research into N-SMase activity in cell culture and various tissues, the lack, until recently, of molecular identification of specific N-SMase enzymes had precluded specific insights into the regulation, physiological, and pathological roles of these proteins. In this review, we summarize, for the first time, current knowledge of the N-SMase family, focusing on cloned members from bacteria, yeast, and mammalian cells. We also briefly consider the major future directions for N-SMase research which promises highly significant and specific insight into sphingolipid-mediated functions.     

Neutral sphingomyelinases and nSMase2: bridging the gaps

There is strong evidence indicating a role for ceramide as a second messenger in processes such as apoptosis, cell growth and differentiation, and cellular responses to stress. Ceramide formation from the hydrolysis of sphingomyelin is considered to be a major pathway of stress-induced ceramide production with magnesium-dependent neutral sphingomyelinase (N-SMase) identified as a prime candidate in this pathway. The recent cloning of a mammalian N-SMase-nSMase2- and generation of nSMase2 knockout/mutant mice have now provided vital tools with which to further study the regulation and roles of this enzyme in both a physiological and pathological context. In the present review, we summarize current knowledge on N-SMase relating this to what is known about nSMase2. We also discuss the future areas of nSMase2 research important for molecular understanding of this enzyme and its physiological roles.     

Neutral sphingomyelinases and nSMase2: Bridging the gaps

There is strong evidence indicating a role for ceramide as a second messenger in processes such as apoptosis, cell growth and differentiation, and cellular responses to stress. Ceramide formation from the hydrolysis of sphingomyelin is considered to be a major pathway of stress-induced ceramide production with magnesium-dependent neutral sphingomyelinase (N-SMase) identified as a prime candidate in this pathway. The recent cloning of a mammalian N-SMase-nSMase2- and generation of nSMase2 knockout/mutant mice have now provided vital tools with which to further study the regulation and roles of this enzyme in both a physiological and pathological context. In the present review, we summarize current knowledge on N-SMase relating this to what is known about nSMase2. We also discuss the future areas of nSMase2 research important for molecular understanding of this enzyme and its physiological roles.     

Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production. However, there is little information on the physiological regulation and roles of the cloned N-SMase enzyme, nSMase2. In this study, nSMase2 was found to translocate acutely to the plasma membrane of A549 epithelial cells in response to tumor necrosis factor alpha (TNF-alpha) in a time- and dose-dependent manner. Additionally, TNF-alpha increased N-SMase activity rapidly and transiently both endogenously and in cells overexpressing nSMase2. Furthermore, the translocation of nSMase2 was regulated by p38-alpha MAPK, but not ERK or JNK, and the increase in endogenous N-SMase activity was abrogated by p38 MAPK inhibition. In addition, both p38-alpha MAPK and nSMase2 were implicated in the TNF-alpha-stimulated up-regulation of the adhesion proteins vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM), but this was largely independent of NF-kappaB activation. These data reveal p38 MAPK as an upstream regulator of nSMase2 and indicate a role for nSMase2 in pro-inflammatory responses induced by TNF-alpha as a regulator of adhesion proteins.     

The Roles of Neutral Sphingomyelinases in Neurological Pathologies

The neutral sphingomyelinases (N-SMases) are a group of Mg²⁺-dependent enzymes with a pH optimum in the neutral range. N-SMases catalyze the conversion of sphingomyelin to ceramide and have been found particularly enriched in brain tissue. N-SMase activity has been implicated in many physiological and pathological processes affecting the brain and nervous system. In this review, we discuss the proposed functions of N-SMase with a particular emphasis on its role in neurological disorders, such as age-related neurodegeneration, Alzheimer’s disease, HIV-associated dementia, atherosclerosis, ischemia–reperfusion injury, and cancer.     

Molecular modeling of human neutral sphingomyelinase provides insight into its molecular interactions

The neutral sphingomyelinase (N-SMase) is considered a major candidate for mediating the stress-induced production of ceramide, and it plays an important role in cell-cycle arrest, apoptosis, inflammation, and eukaryotic stress responses. Recent studies have identified a small region at the very N-terminus of the 55 kDa tumour necrosis factor receptor (TNF-R55), designated the neutral sphingomyelinase activating domain (NSD) that is responsible for the TNF-induced activation of N-SMase. There is no direct association between TNF-R55 NSD and N-SMase; instead, a protein named factor associated with N-SMase activation (FAN) has been reported to couple the TNF-R55 NSD to N-SMase. Since the three-dimensional fold of N-SMase is still unknown, we have modeled the structure using the protein fold recognition and threading method. Moreover, we propose models for the TNF-R55 NSD as well as the FAN protein in order to study the structural basis of N-SMase activation and regulation. Protein-protein interaction studies suggest that FAN is crucially involved in mediating TNF-induced activation of the N-SMase pathway, which in turn regulates mitogenic and proinflammatory responses. Inhibition of N-SMase may lead to reduction of ceramide levels and hence may provide a novel therapeutic strategy for inflammation and autoimmune diseases. Molecular dynamics (MD) simulations were performed to check the stability of the predicted model and protein-protein complex; indeed, stable RMS deviations were obtained throughout the simulation. Furthermore, in silico docking of low molecular mass ligands into the active site of N-SMase suggests that His135, Glu48, Asp177, and Asn179 residues play crucial roles in this interaction. Based on our results, these ligands are proposed to be potent and selective N-SMase inhibitors, which may ultimately prove useful as lead compounds for drug development.     

The Akt kinases: Isoform specificity in metabolism and cancer

The Akt (PKB) protein kinases are critical regulators of human physiology that control an impressive array of diverse cellular functions, including the modulation of growth, survival, proliferation and metabolism. The Akt kinase family is comprised of three highly homologous isoforms: Akt1 (PKBα), Akt2 (PKBβ) and Akt3 (PKBγ). Phenotypic analyses of Akt isoform knockout mice documented Akt isoform specific functions in the regulation of cellular growth, glucose homeostasis and neuronal development. Those studies establish that the functions of the different Akt kinases are not completely overlapping and that isoform-specific signaling contributes to the diversity of Akt activities. However, despite these important advances, a thorough understanding about the specific roles of Akt family members and the molecular mechanisms that determine Akt isoform functional specificity will be essential to elucidate the complexity of Akt regulated cellular processes and how Akt isoform-specific deregulation might contribute to disease states. Here, we summarize recent advances in understanding the roles of Akt isoforms in the regulation of metabolism and cancer, and possible mechanisms contributing to Akt isoform functional specificity.     

Identification and Characterization of Murine Mitochondria-associated Neutral Sphingomyelinase (MA-nSMase), the Mammalian Sphingomyelin Phosphodiesterase 5

Sphingolipids play important roles in regulating cellular responses. Although mitochondria contain sphingolipids, direct regulation of their levels in mitochondria or mitochondria-associated membranes is mostly unclear. Neutral SMase (N-SMase) isoforms, which catalyze hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine, have been found in the mitochondria of yeast and zebrafish, yet their existence in mammalian mitochondria remains unknown. Here, we have identified and cloned a cDNA based on nSMase homologous sequences. This cDNA encodes a novel protein of 483 amino acids that displays significant homology to nSMase2 and possesses the same catalytic core residues as members of the extended N-SMase family. A transiently expressed V5-tagged protein co-localized with both mitochondria and endoplasmic reticulum markers in MCF-7 and HEK293 cells; accordingly, the enzyme is referred to as mitochondria-associated nSMase (MA-nSMase). MA-nSMase was highly expressed in testis, pancreas, epididymis, and brain. MA-nSMase had an absolute requirement for cations such as Mg²⁺ and Mn²⁺ and activation by the anionic phospholipids, especially phosphatidylserine and the mitochondrial cardiolipin. Importantly, overexpression of MA-nSMase in HEK293 cells significantly increased in vitro N-SMase activity and also modulated the levels of SM and ceramide, indicating that the identified cDNA encodes a functional SMase. Thus, these studies identify and characterize, for the first time, a mammalian MA-nSMase. The characterization of MA-nSMase described here will contribute to our understanding of pathways regulated by sphingolipid metabolites, particularly with reference to the mitochondria and associated organelles.     

Yeast as a model to study apoptosis?

Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms.The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions. Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, . . .) found in mammals. This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis.Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics).This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.     

Autophagic processes in plants: mechanisms, regulation and function

Large numbers of publications investigating the molecular details, the regulation and the physiological roles of autophagic processes have appeared over the last few years, dealing with animals, plants and unicellular eukaryotic organisms. This strong interest is caused by the fact that autophagic processes are ubiquitous in eukaryotic organisms. They are involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. This review article aims to describe the discovery of autophagy, the molecular details of this complex process, its regulation, and its specific functions in plants.     

NOX5: from basic biology to signaling and disease

In mammals, the NADPH oxidase family of enzymes comprises seven members: NOXs 1-5, DUOX1, and DUOX2. All of these enzymes function to move an electron across cellular membranes, transferring it to oxygen to generate the superoxide anion. This generation of reactive oxygen species has important physiological and pathophysiological roles. NOX5 is perhaps the least well understood of these NOX isoforms, in part because the gene is not present in mice or rats. In recent years, however, there has been a rapid increase in our understanding of the NOX5 gene, the structural and biochemical aspects of the NOX5 enzyme, the role NOX5 plays in health and disease, and the development of novel NOX inhibitors. This review takes a look back at some historical aspects of the discovery of NOX5 and summarizes our current understanding of the enzyme.     

Phospholipase D- and phosphatidic acid-mediated signaling in plants

The phospholipase D (PLD) family in higher plants is composed of multiple members, and each of the Arabidopsis PLDs characterized displays distinguishable properties in activity regulation and/or lipid preferences. The molecular and biochemical heterogeneities of the plant PLDs play important roles in the timing, location, and amount of phosphatidic acid (PA) produced. PLD-catalyzed production of PA has been shown to play important roles in plant growth, development, and response to various stresses, including drought, salinity, freezing, and nutrient deficiency. PLD and PA affect cellular processes through different modes of action, including direct target protein binding and biophysical effects on cell membranes. Improved knowledge on the mechanism by which specific PLDs and PA mediate given plant responses will facilitate the understanding of the molecular processes that connect the stimulus perception on membranes to intracellular actions and physiological responses.     

Functional Surfaces on the Actin-binding Protein Coronin Revealed by Systematic Mutagenesis

Coronin is a conserved actin-binding protein that co-functions with ADF/cofilin and Arp2/3 complex to govern cellular actin dynamics. Despite emerging roles for coronin in a range of physiological processes and disease states, a detailed understanding of the molecular interactions of coronin with actin and other binding partners has been lacking. Here, we performed a systematic mutational analysis of surfaces on the yeast coronin β-propeller domain, which binds to F-actin and is conserved in all coronin family members. We generated 21 mutant alleles and analyzed their biochemical effects on actin binding and ADF/cofilin activity. Conserved actin-binding residues mapped to a discrete ridge stretching across one side of the β-propeller. Mutants defective in actin binding showed loss of synergy with ADF/cofilin in severing filaments, diminished localization to actin structures in vivo, and loss of coronin overexpression growth defects. In addition, one allele showed normal actin binding but impaired functional interactions with ADF/cofilin. Another allele showed normal actin binding but failed to cause coronin overexpression defects. Together, these results indicate that actin binding is critical for many of the biochemical and cellular functions of coronin and that the β-propeller domain mediates additional functional interactions with ADF/cofilin and possibly other ligands. Conservation of the actin-binding surfaces across distant species and in all three major classes of coronin isoforms suggests that the nature of the coronin-actin association may be similar in other family members.     

Biology and Biochemistry of Plant Phospholipases

Phospholipases are a complex group of enzymes that hydrolyze phospholipids. The plant phospholipase family is composed of multiple members with varying positional specificity, and each type is represented by multiple isoforms distinguishable by their structural, catalytic, and physiological characteristics. A large number of phospholipase genes and gene families have been identified and the biochemical properties of several members have been characterized, revealing considerable molecular and catalytic diversity. Forward and reverse genetics has further revealed that phospholipases are widely involved in physiological processes including lipid metabolism, cell signaling, and responses to biotic and abiotic stresses. Such studies have highlighted the potential biotechnological value of phospholipases as targets for improving stress tolerance. The catalytic diversity of various phospholipase isoforms is also of increasing interest for industrial biocatalysis. This review focuses on recently acquired information on biochemical, molecular and functional aspects of plant phospholipases.     

Physiological roles for G protein-regulated adenylyl cyclase isoforms: insights from knockout and overexpression studies

Cyclic AMP is a universal second messenger, produced by a family of adenylyl cyclase (AC) enzymes. The last three decades have brought a wealth of new information about the regulation of cyclic AMP production by ACs. Nine hormone-sensitive, membrane-bound AC isoforms have been identified in addition to a tenth isoform that lacks membrane spans and more closely resembles the cyanobacterial AC enzymes. New model systems for purifying and characterizing the catalytic domains of AC have led to the crystal structure of these domains and the mapping of numerous interaction sites. However, big hurdles remain in unraveling the roles of individual AC isoforms and their regulation in physiological systems. In this review we explore the latest on AC knockout and overexpression studies to better understand the roles of G protein regulation of ACs in the brain, olfactory bulb, and heart.     

Casein Kinase I: Spatial Organization and Positioning of a Multifunctional Protein Kinase Family

The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans. Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins. While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. All five S. cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions. New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes. In particular, CKI, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus. Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function. Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events. This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases.     

Role of Rab family GTPases and their effectors in melanosomal logistics

Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.     

Regulation of subcellular localization of alpha1-adrenoceptor subtypes

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the alpha1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with alpha1B-AR in vitro and in vivo through the specific site, and that in cells which co-express alpha1B-AR and gC1q-R, the subcellular localization of alpha1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of alpha1B-ARs.