Biochemical Society Transactions

Advances in the Cellular and Molecular Biology of Angiogenesis

Stuart Egginton and Roy Bicknell — 2011-12-01

Capillaries have been recognized for over a century as one of the most important components in regulating tissue oxygen transport, and their formation or angiogenesis a pivotal element of tissue remodelling during development and adaptation. Clinical interest stems from observations that both excessive and inadequate vascular growth plays a major role in human diseases, and novel developments in treatments for cancer and eye disease increasingly rely on anti-angiogenic therapies. Although the discovery of VEGF (vascular endothelial growth factor) provided the first clue for specificity of signalling in endothelial cell activation, understanding the integrative response that drives angiogenesis requires a much broader perspective. The Advances in the Cellular and Molecular Biology of Angiogenesis meeting brought together researchers at the forefront of this rapidly moving field to provide an update on current understanding, and the most recent insights into molecular and cellular mechanisms of vascular growth. The plenary lecture highlighted the integrative nature of the angiogenic process, whereas invited contributions from basic and clinician scientists described fundamental mechanisms and disease-associated issues of blood vessel formation, grouped under a number of themes to aid discussion. These articles will appeal to academic, clinical and pharmaceutical scientists interested in the molecular and cellular basis of angiogenesis, their modulation or dysfunction in human diseases, and application of these findings towards translational medicine.

The critical role of VEGF in skeletal muscle angiogenesis and blood flow

Peter D. Wagner — 2011-12-01

VEGF (vascular endothelial growth factor) is well known as an important molecule in angiogenesis. Its inhibition is pursued as an anticancer therapy; its enhancement as therapy for tissue ischaemia. In the present paper, its role in skeletal muscle is explored, both at rest and after exercise. Muscle VEGF mRNA and protein are increased severalfold after heavy exercise. Whereas global VEGF knockout is embryonically lethal, muscle-specific knockout is not, providing models for studying its functional significance. Its deletion in adult mouse skeletal muscle: (i) reduces muscle capillarity by more than 50%, (ii) decreases exercise endurance time by approximately 80%, and (iii) abolishes the angiogenic response to exercise training. What causes VEGF to increase with exercise is not clear. Despite regulation by HIF (hypoxia-inducible factor), increased HIF on exercise, and PO₂ falling to single digit values during exercise, muscle-specific HIF knockout does not impair performance or capillarity, leaving many unanswered questions.

Inhibition of angiogenesis and the angiogenesis/invasion shift

2011-12-01

Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes up-regulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that anti-angiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools.

Class 3 semaphorins and their receptors in physiological and pathological angiogenesis

Carolyn A. Staton — 2011-12-01

Class 3 semaphorins (Sema3) are a family of secreted proteins that were originally identified as axon guidance factors mediating their signal transduction by forming complexes with neuropilins and plexins. However, the wide expression pattern of Sema3 suggested additional functions other than those associated with the nervous system, and indeed many studies have now indicated that Sema3 proteins and their receptors play a role in angiogenesis. The present review specifically focuses on recent evidence for this role in both physiological and pathological angiogenesis.

Shear stress, tip cells and regulators of endothelial migration

Xiaodong Zhuang, Darren Cross, Victoria L. Heath and Roy Bicknell — 2011-12-01

We have in recent years described several endothelial-specific genes that mediate cell migration. These include Robo4 (roundabout 4), CLEC14A (C-type lectin 14A) and ECSCR (endothelial cell-specific chemotaxis regulator) [formerly known as ECSM2 (endothelial cell-specific molecule 2)]. Loss of laminar shear stress induces Robo4 and CLEC14A expression and an endothelial 'tip cell' phenotype. Low shear stress is found not only at sites of vascular occlusion such as thrombosis and embolism, but also in the poorly structured vessels that populate solid tumours. The latter probably accounts for strong expression of Robo4 and CLEC14A on tumour vessels. The function of Robo4 has, in the past, aroused controversy. However, the recent identification of Unc5B as a Robo4 ligand has increased our understanding and we hypothesize that Robo4 function is context-dependent. ECSCR is another endothelial-specific protein that promotes filopodia formation and migration, but, in this case, expression is independent of shear stress. We discuss recent papers describing ECSCR, including intracellular signalling pathways, and briefly contrast these with signalling by Robo4.

An unexpected tail of VEGF and PlGF in pre-eclampsia

David O. Bates — 2011-12-01

PET (pre-eclamptic toxaemia), characterized by pregnancy-related hypertension and proteinuria, due to widespread endothelial dysfunction, is a primary cause of maternal morbidity. Altered circulating factors, particularly the VEGF (vascular endothelial growth factor) family of proteins and their receptors, are thought to be key contributors to this disease. Plasma from patients with PET induces numerous cellular and physiological changes in endothelial cells, indicating the presence of a circulating imbalance of the normal plasma constituents. These have been narrowed down to macromolecules of the VEGF family of proteins and receptors. It has been shown that responses of endothelial cells in intact vessels to plasma from patients with pre-eclampsia is VEGF-dependent. It has recently been shown that this may be specific to the VEGF₁₆₅b isoform, and blocked by addition of recombinant human PlGF (placental growth factor). Taken together with results that show that sVEGFR1 (soluble VEGF receptor 1) levels are insufficient to bind VEGF-A in human plasma from patients with pre-eclampsia, and that other circulating macromolecules bind, but do not inactivate, VEGF-A, this suggests that novel hypotheses involving altered bioavailability of VEGF isoforms resulting from reduced or bound PlGF, or increased sVEGFR1 increasing biological activity of circulating plasma, could be tested. This suggests that knowing how to alter the balance of VEGF family members could prevent endothelial activation, and potentially some symptoms, of pre-eclampsia.

How neuropilin-1 regulates receptor tyrosine kinase signalling: the knowns and known unknowns

Ian C. Zachary — 2011-12-01

Essential roles of NRP1 (neuropilin-1) in cardiovascular development and in neuronal axon targeting during embryogenesis are thought to be mediated primarily through binding of NRP1 to two unrelated types of ligands: the VEGF (vascular endothelial growth factor) family of angiogenic cytokines in the endothelium, and the class 3 semaphorins in neurons. A widely accepted mechanism for the role of NRP1 in the endothelium is VEGF binding to NRP1 and VEGFR2 (VEGF receptor 2) and VEGF-dependent formation of complexes or NRP1-VEGFR2 holoreceptors with enhanced signalling activity and biological function. However, although some basic features of this model are solidly based on biochemical and cellular data, others are open to question. Furthermore, a mechanistic account of NRP1 has to accommodate research which emphasizes the diversity of NRP1 functions in different cell types and particularly an emerging role in signalling by other growth factor ligands for RTKs (receptor tyrosine kinases) such as HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). It is uncertain, however, whether the model of NRP1-RTK heterocomplex formation applies in all of these situations. In the light of these developments, the need to explain mechanistically the role of NRP1 in signalling is coming increasingly to the fore. The present article focuses on some of the most important unresolved questions concerning the mechanism(s) through which NRP1 acts, and highlights recent findings which are beginning to generate insights into these questions.

Molecular control of angiopoietin signalling

2011-12-01

The angiopoietins act through the endothelial receptor tyrosine kinase Tie2 to regulate vessel maturation in angiogenesis and control quiescence and stability of established vessels. The activating ligand, Ang1 (angiopoietin-1), is constitutively expressed by perivascular cells, and the ability of endothelial cells to respond to the ligand is controlled at the level of the Ang1 receptor. This receptor interacts with the related protein Tie1 on the cell surface, and Tie1 inhibits Ang1 signalling through Tie2. The responsiveness of endothelium to Ang1 is determined by the relative levels of Tie2 and the inhibitory co-receptor Tie1 in the cells. Tie1 undergoes regulated ectodomain cleavage which is stimulated by a range of factors including VEGF (vascular endothelial growth factor), inflammatory cytokines and changes in shear stress. Ectodomain cleavage of Tie1 relieves inhibition of Tie2 and enhances Ang1 signalling. This mechanism regulates Ang1 signalling without requiring changes in the level of the ligand and allows Ang1 signalling to be co-ordinated with other signals in the cellular environment. Regulation of signalling at the level of receptor responsiveness may be an important adaptation in systems in which an activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.

Uses of the in vitro endothelial-fibroblast organotypic co-culture assay in angiogenesis research

Clare Hetheridge, Georgia Mavria and Harry Mellor — 2011-12-01

Angiogenesis is a complex process that involves multiple cellular events. In addition to receiving inputs from a range of stimulatory and inhibitory factors, endothelial cells undergoing angiogenesis make multiple interactions with the extracellular matrix and with other cell types in the stroma. Recreating angiogenesis in vitro is probably an impossible goal; however, a number of assays have been developed that recapitulate many of the key events of the process. These assays are indispensible tools for investigating the signalling pathways that control the formation of new blood vessels. In the present paper, we review the organotypic co-culture assay of angiogenesis - until recently, a comparatively underemployed assay, but one with a number of powerful advantages for angiogenesis research. We give a set of optimized protocols for its use, including protocols for siRNA (small interfering RNA)-based screens, and we discuss appropriate methods for obtaining quantitative data from the assay.

PPARs and angiogenesis

David Bishop‑Bailey — 2011-12-01

The PPAR (peroxisome-proliferator-activated receptor) family consists of three ligand-activated nuclear receptors: PPARα, PPARβ/δ and PPARγ. These PPARs have important roles in the regulation of glucose and fatty acid metabolism, cell differentiation and immune function, but were also found to be expressed in endothelial cells in the late 1990s. The early endothelial focus of PPARs was PPARγ, the molecular target for the insulin-sensitizing thiazolidinedione/glitazone class of drugs. Activation of PPARγ was shown to inhibit angiogenesis in vitro and in models of retinopathy and cancer, whereas more recent data point to a critical role in the development of the vasculature in the placenta. Similarly, PPARα, the molecular target for the fibrate class of drugs, also has anti-angiogenic properties in experimental models. In contrast, unlike PPARα or PPARγ, activation of PPARβ/δ induces angiogenesis, in vitro and in vivo, and has been suggested to be a critical component of the angiogenic switch in pancreatic cancer. Moreover, PPARβ/δ is an exercise mimetic and appears to contribute to the angiogenic remodelling of cardiac and skeletal muscle induced by exercise. This evidence and the emerging mechanisms by which PPARs act in endothelial cells are discussed in more detail.

The role of RhoJ in endothelial cell biology and angiogenesis

2011-12-01

RhoJ is an endothelially expressed member of the Cdc42 (cell division cycle 42) subfamily of small Rho GTPases. It is expressed in both the developing mammalian vasculature and the vascular beds of a number of adult tissues, with its expression regulated by the endothelial transcription factor ERG (ETS-related gene). RhoJ has been shown to regulate endothelial motility, tubulogenesis and lumen formation in vitro, and modulates the vascularization of Matrigel plugs in vivo. Both vascular endothelial growth factor and semaphorin 3E have been found to affect its activation. RhoJ has been shown to be a focal-adhesion-localized Rho GTPase which can modulate focal adhesion number, actomyosin contractility and activity of Cdc42 and Rac1. The present review discusses the biology of RhoJ with a focus on recent reports of its role in endothelial cells and angiogenesis.

New pathways and mechanisms regulating and responding to Delta-like ligand 4-Notch signalling in tumour angiogenesis

Chern Ein Oon and Adrian L. Harris — 2011-12-01

Notch signalling is a key pathway controlling angiogenesis in normal tissues and tumours. This has become a major focus of development of anticancer therapy, but to develop this appropriately, we need further understanding of the mechanisms of regulation of Dll4 (Delta-like ligand 4), a key endothelial Notch ligand. Dll4 and VEGF (vascular endothelial growth factor) cross-talk, with VEGF up-regulation of Dll4 and Dll4 down-regulating VEGFR (VEGF receptor) signalling. Both are essential for normal angiogenesis, and blockade of one may produce compensatory changes in the other. The present review considers recent developments in the regulation of Dll4 expression and functions, its role as a mechanism of resistance to anti-angiogenic therapy, and methods needed to develop effective therapy against this target.

Is inflammation the cause of pre-eclampsia?

Wenda Ramma and Asif Ahmed — 2011-12-01

It has been proposed that either excessive inflammation or an imbalance in angiogenic factors cause pre-eclampsia. In the present review, the arguments for and against the role of inflammation and/or angiogenic imbalance as the cause of pre-eclampsia are discussed on the basis of the Bradford-Hill criteria for disease causation. Although both angiogenic imbalance and systemic inflammation are implicated in pre-eclampsia, the absence of temporality of inflammatory markers with pre-eclampsia challenges the concept that excessive inflammation is the cause of pre-eclampsia. In contrast, the elevation of anti-angiogenic factors that precede the clinical signs of pre-eclampsia fulfils the criterion of temporality. The second most important criterion is the dose-response relationship. Although such a relationship has not been proven between pro-inflammatory cytokines and pre-eclampsia, high levels of anti-angiogenic factors have been shown to correlate with increased incidence and disease severity, hence satisfying this condition. Finally, as the removal of circulating sFlt-1 (soluble Fms-like tyrosine kinase receptor-1) from pre-eclamptic patients significantly improves the clinical outcome, it fulfils the Hill's experiment principle, which states that removal of the cause by an appropriate experimental regimen should ameliorate the condition. In contrast, treatment with high doses of corticosteroid fails to improve maternal outcome in pre-eclampsia, despite suppressing inflammation. Inflammation may enhance the pathology induced by the imbalance in the angiogenic factors, but does not by itself cause pre-eclampsia. Development of therapies based on the angiogenic and cytoprotective mechanisms seems more promising.

Vascular remodelling in human skeletal muscle

Thomas Gustafsson — 2011-12-01

Exercise-induced angiogenesis in skeletal muscle involves both non-sprouting and sprouting angiogenesis and results from the integrated responses of multiple systems and stimuli. VEGF-A (vascular endothelial growth factor A) levels are increased in exercised muscle and have been demonstrated to be critical for exercise-induced capillary growth. Only limited information is available regarding the role of other angiogenic and angiostatic factors in exercise, but changes in the angiopoietin family following repetitive bouts of exercise occur in a pattern that is favourable for angiogenesis. Results from other angiogenic model systems, indicate that miRNAs (microRNAs) are important factors in the regulation of angiogenesis and thus to explore their role as regulators of exercise induced angiogenesis will be an important avenue of study in the future. ECM (extracellular matrix) remodelling and activation of MMPs (matrix metalloproteinases) are, to some extent, overlooked players in skeletal muscle adaptation. Degradation of ECM proteins liberates angiogenic factors from immobilized matrix stores and make cell migration possible. In fact, it is known that MMPs become activated by a single bout of exercise in humans, rapid interstitial changes occur long before any changes in gene transcription could result in protein synthesis and inhibition of MMP activity completely abolishes sprouting angiogenesis. A growing body of evidence suggests that circulating and resident progenitor cells, in addition to other cell types located in skeletal muscle tissue, participate in skeletal muscle angiogenesis by various mechanisms. However, more studies are needed before these can be confirmed as mechanisms of exercise-induced capillary growth.

In vivo shear stress response

Stuart Egginton — 2011-12-01

EC (endothelial cell) responses to shear stress generated by vascular perfusion play an important role in circulatory homoeostasis, whereas abnormal responses are implicated in vascular diseases such as hypertension and atherosclerosis. ECs subjected to high shear stress in vitro alter their morphology, function and gene expression. The molecular basis for mechanotransduction of a shear stress signal, and the identity of the sensing mechanisms, remain unclear with many candidates under investigation. Translating these findings in vivo has proved difficult. The role of VEGF (vascular endothelial growth factor) flow-dependent nitric oxide release in remodelling skeletal muscle microcirculation is established for elevated (activity, dilatation) and reduced (overload, ischaemia) shear stress, although their temporal relationship to angiogenesis varies. It is clear that growth factor levels may offer only a permissive environment, and alteration of receptor levels may be a viable therapeutic target. Angiogenesis in vivo appears to be a graded phenomenon, and capillary regression on withdrawal of stimulus may be rapid. Combinations of physiological angiogenic stimuli appear not to be additive.

New insights into the plasticity of the endothelial phenotype

Lindsay S. Cooley and Dylan R. Edwards — 2011-12-01

The mammalian vascular system consists of two distinct, but closely related, networks: the blood vasculature (itself divided into arterial and venous networks) and the lymphatic vasculature. EC (endothelial cell) lineage specification has been proposed to be determined during embryonic development, after which the ECs are committed to their fate. However, increasing evidence suggests that ECs retain various degrees of plasticity, and have the ability to express characteristics of alternative cell lineages. Therapeutic control of endothelial plasticity will allow greater understanding of the genesis and treatment of several vascular diseases.

To sprout or to split? VEGF, Notch and vascular morphogenesis

Roberto Gianni‑Barrera, Marianna Trani, Silvia Reginato and Andrea Banfi — 2011-12-01

Therapeutic angiogenesis is an attractive strategy to treat patients suffering from peripheral or coronary artery disease. VEGF (vascular endothelial growth factor-A) is the fundamental factor controlling vascular growth in both development and postnatal life. The interplay between the VEGF and Notch signalling pathway has been recently found to regulate the morphogenic events leading to the growth of new vessels by sprouting. Angiogenesis can also take place by an alternative process, i.e. intussusception or vascular splitting. However, little is known about its role in therapeutic angiogenesis and its molecular regulation. In the present article, we briefly review how VEGF dose determines the induction of normal or aberrant angiogenesis and the molecular regulation of sprouting angiogenesis by Notch signalling, and compare this process with intussusception.

Angiogenic properties of the chemokine RANTES/CCL5

2011-12-01

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.

Taming of the wild vessel: promoting vessel stabilization for safe therapeutic angiogenesis

Silvia Reginato, Roberto Gianni‑Barrera and Andrea Banfi — 2011-12-01

VEGF (vascular endothelial growth factor) is the master regulator of blood vessel growth. However, it displayed substantial limitations when delivered as a single gene to restore blood flow in ischaemic conditions. Indeed, uncontrolled VEGF expression can easily induce aberrant vascular structures, and short-term expression leads to unstable vessels. Targeting the second stage of the angiogenic process, i.e. vascular maturation, is an attractive strategy to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations of VEGF-based gene therapy, briefly summarizes the current knowledge of the molecular and cellular regulation of vascular maturation, and describes recent pre-clinical evidence on how the maturation stage could be targeted to achieve therapeutic angiogenesis.

Angiogenesis regulation by TGFbeta signalling: clues from an inherited vascular disease

Marwa Mahmoud, Paul D. Upton and Helen M. Arthur — 2011-12-01

Studies of rare genetic diseases frequently reveal genes that are fundamental to life, and the familial vascular disorder HHT (hereditary haemorrhagic telangiectasia) is no exception. The majority of HHT patients are heterozygous for mutations in either the ENG (endoglin) or the ACVRL1 (activin receptor-like kinase 1) gene. Both genes are essential for angiogenesis during development and mice that are homozygous for mutations in Eng or Acvrl1 die in mid-gestation from vascular defects. Recent development of conditional mouse models in which the Eng or Acvrl1 gene can be depleted in later life have confirmed the importance of both genes in angiogenesis and in the maintenance of a normal vasculature. Endoglin protein is a co-receptor and ACVRL1 is a signalling receptor, both of which are expressed primarily in endothelial cells to regulate TGFβ (transforming growth factor β) signalling in the cardiovasculature. The role of ACVRL1 and endoglin in TGFβ signalling during angiogenesis is now becoming clearer as interactions between these receptors and additional ligands of the TGFβ superfamily, as well as synergistic relationships with other signalling pathways, are being uncovered. The present review aims to place these recent findings into the context of a better understanding of HHT and to summarize recent evidence that confirms the importance of endoglin and ACVRL1 in maintaining normal cardiovascular health.

The emerging role of tetraspanin microdomains on endothelial cells

2011-12-01

Tetraspanins function as organizers of the cell surface by recruiting specific partner proteins into tetraspanin-enriched microdomains, which regulate processes such as cell adhesion, signalling and intracellular trafficking. Endothelial cells appear to express at least 23 of the 33 human tetraspanins, and a number of recent studies have demonstrated their importance in endothelial cell biology. Tetraspanin CD151 is essential for pathological angiogenesis, which may in part be due to regulation of its main partner proteins, the laminin-binding integrins α3β1, α6β1 and α6β4. CD9 and CD151 are essential for leucocyte recruitment during an inflammatory response, through the formation of pre-assembled nano-platforms containing the adhesion molecules ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1), which ultimately coalesce to form docking structures around captured leucocytes. Tetraspanin CD63 also facilitates leucocyte capture by promoting clustering of the adhesion molecule P-selectin. Finally, Tspan12 is required for blood vessel development in the eye, through regulation of Norrin-induced Frizzled-4 signalling, such that Tspan12 mutations can lead to human disease. Future studies on these and other endothelial tetraspanins are likely to provide further novel insights into angiogenesis and inflammation.

Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse

2011-12-01

Lymphatic vessels, the second vascular system of higher vertebrates, are indispensable for fluid tissue homoeostasis, dietary fat resorption and immune surveillance. Not only are lymphatic vessels formed during fetal development, when the lymphatic endothelium differentiates and separates from blood endothelial cells, but also lymphangiogenesis occurs during adult life under conditions of inflammation, wound healing and tumour formation. Under all of these conditions, haemopoietic cells can exert instructive influences on lymph vessel growth and are essential for the vital separation of blood and lymphatic vessels. LECs (lymphatic endothelial cells) are characterized by expression of a number of unique genes that distinguish them from blood endothelium and can be utilized to drive reporter genes in a lymph endothelial-specific fashion. In the present paper, we describe the Prox1 (prospero homeobox protein 1) promoter-driven expression of the fluorescent protein mOrange2, which allows the specific intravital visualization of lymph vessel growth and behaviour during mouse fetal development and in adult mice.

Nuclear Envelope Disease and Chromatin Organization

Juliet A. Ellis and Sue Shackleton — 2011-12-01

The fifth U.K. meeting on nuclear envelope disease and chromatin brought together international experts from across the field of nuclear envelope biology to discuss the advancements in a class of tissue-specific degenerative diseases called the laminopathies. Clinically, these range from relatively mild fat-wasting disorders to the severe premature aging condition known as Hutchinson-Gilford progeria syndrome. Since the first association of the nuclear envelope with human inherited disease in 1994, there has been an exponential increase in an unexpected variety of functions associated with nuclear envelope proteins, ranging from mechanical support and nucleocytoskeletal connections to regulation of chromatin organization and gene expression. This Biochemical Society Focused Meeting reinforced the functional complexity of nuclear-associated diseases, revealed new avenues to be investigated and highlighted the signalling pathways suitable as therapeutic targets.

Clinical and genetic heterogeneity in laminopathies

Anne T. Bertrand, Khadija Chikhaoui, Rabah Ben Yaou and Gisèle Bonne — 2011-12-01

Mutations in the LMNA gene encoding lamins A/C are responsible for more than ten different disorders called laminopathies which affect various tissues in an isolated (striated muscle, adipose tissue or peripheral nerve) or systemic (premature aging syndromes) fashion. Overlapping phenotypes are also observed. Associated with this wide clinical variability, there is also a large genetic heterogeneity, with 408 different mutations being reported to date. Whereas a few hotspot mutations emerge for some types of laminopathies, relationships between genotypes and phenotypes remain poor for laminopathies affecting the striated muscles. In addition, there is important intrafamilial variability, explained only in a few cases by digenism, thus suggesting an additional contribution from modifier genes. In this regard, a chromosomal region linked to the variability in the age at onset of myopathic symptoms in striated muscle laminopathies has recently been identified. This locus is currently under investigation to identify modifier variants responsible for this variability.

The LINC complex and human disease

Peter Meinke, Thuy Duong Nguyen and Manfred S. Wehnert — 2011-12-01

The LINC (linker of nucleoskeleton and cytoskeleton) complex is a proposed mechanical link tethering the nucleo- and cyto-skeleton via the NE (nuclear envelope). The LINC components emerin, lamin A/C, SUN1, SUN2, nesprin-1 and nesprin-2 interact with each other at the NE and also with other binding partners including actin filaments and B-type lamins. Besides the mechanostructural functions, the LINC complex is also involved in signalling pathways and gene regulation. Emerin was the first LINC component associated with a human disease, namely EDMD (Emery-Dreifuss muscular dystrophy). Later on, other components of the LINC complex, such as lamins A/C and small isoforms of nesprin-1 and nesprin-2, were found to be associated with EDMD, reflecting a genetic heterogeneity that has not been resolved so far. Only approximately 46% of the EDMD patients can be linked to genes of LINC and non-LINC components, pointing to further genes involved in the pathology of EDMD. Obvious candidates are the LINC proteins SUN1 and SUN2. Recently, screening of binding partners of LINC components as candidates identified LUMA (TMEM43), encoding a binding partner of emerin and lamins, as a gene involved in atypical EDMD. Nevertheless, such mutations contribute only to a very small fraction of EDMD patients. EDMD-causing mutations in STA/EMD (encoding emerin) that disrupt emerin binding to Btf (Bcl-2-associated transcription factor), GCL (germ cell-less) and BAF (barrier to autointegration factor) provide the first glimpses into LINC being involved in gene regulation and thus opening new avenues for functional studies. Thus the association of LINC with human disease provides tools for understanding its functions within the cell.

Prelamin A-mediated nuclear envelope dynamics in normal and laminopathic cells

Giovanna Lattanzi — 2011-12-01

Prelamin A is the precursor protein of lamin A, a major constituent of the nuclear lamina in higher eukaryotes. Increasing attention to prelamin A processing and function has been given after the discovery, from 2002 to 2004, of diseases caused by prelamin A accumulation. These diseases, belonging to the group of laminopathies and mostly featuring LMNA mutations, are characterized, at the clinical level, by different degrees of accelerated aging, and adipose tissue, skin and bone abnormalities. The outcome of studies conducted in the last few years consists of three major findings. First, prelamin A is processed at different rates under physiological conditions depending on the differentiation state of the cell. This means that, for instance, in muscle cells, prelamin A itself plays a biological role, besides production of mature lamin A. Secondly, prelamin A post-translational modifications give rise to different processing intermediates, which elicit different effects in the nucleus, mostly by modification of the chromatin arrangement. Thirdly, there is a threshold of toxicity, especially of the farnesylated form of prelamin A, whose accumulation is obviously linked to cell and organism senescence. The present review is focused on prelamin A-mediated nuclear envelope modifications that are upstream of chromatin dynamics and gene expression mechanisms regulated by the lamin A precursor.

The nuclear lamina and heterochromatin: a complex relationship

Erin M. Bank and Yosef Gruenbaum — 2011-12-01

In metazoan cells, the heterochromatin is generally localized at the nuclear periphery, whereas active genes are preferentially found in the nuclear interior. In the present paper, we review current evidence showing that components of the nuclear lamina interact directly with heterochromatin, which implicates the nuclear lamina in a mechanism of specific gene retention at the nuclear periphery and release to the nuclear interior upon gene activation. We also discuss recent data showing that mutations in lamin proteins affect gene positioning and expression, providing a potential mechanism for how these mutations lead to tissue-specific diseases.

Cell autonomous and systemic factors in progeria development

2011-12-01

Progeroid laminopathies are accelerated aging syndromes caused by defects in nuclear envelope proteins. Accordingly, mutations in the LMNA gene and functionally related genes have been described to cause HGPS (Hutchinson-Gilford progeria syndrome), MAD (mandibuloacral dysplasia) or RD (restrictive dermopathy). Functional studies with animal and cellular models of these syndromes have facilitated the identification of the molecular alterations and regulatory pathways involved in progeria development. We have recently described a novel regulatory pathway involving miR-29 and p53 tumour suppressor which has provided valuable information on the molecular components orchestrating the response to nuclear damage stress. Furthermore, by using progeroid mice deficient in ZMPSTE24 (zinc metalloprotease STE24 homologue) involved in lamin A maturation, we have demonstrated that, besides these abnormal cellular responses to stress, dysregulation of the somatotropic axis is responsible for some of the alterations associated with progeria. Consistent with these observations, pharmacological restoration of the somatotroph axis in these mice delays the onset of their progeroid features, significantly extending their lifespan and supporting the importance of systemic alterations in progeria progression. Finally, we have very recently identified a novel progeroid syndrome with distinctive features from HGPS and MAD, which we have designated NGPS (Néstor-Guillermo progeria syndrome) (OMIM #614008). This disorder is caused by a mutation in BANF1, a gene encoding a protein with essential functions in the assembly of the nuclear envelope, further illustrating the importance of the nuclear lamina integrity for human health and providing additional support to the study of progeroid syndromes as a valuable source of information on human aging.

The role of DNA damage in laminopathy progeroid syndromes

Christopher J. Hutchison — 2011-12-01

Progeroid laminopathies are characterized by the abnormal processing of lamin A, the appearance of misshapen nuclei, and the accumulation and persistence of DNA damage. In the present article, I consider the contribution of defective DNA damage pathways to the pathology of progeroid laminopathies. Defects in DNA repair pathways appear to be caused by a combination of factors. These include abnormal epigenetic modifications of chromatin that are required to recruit DNA repair pathways to sites of DNA damage, abnormal recruitment of DNA excision repair proteins to sites of DNA double-strand breaks, and unrepairable ROS (reactive oxygen species)-induced DNA damage. At least two of these defective processes offer the potential for novel therapeutic approaches.

Nesprin-3: a versatile connector between the nucleus and the cytoskeleton

Mirjam Ketema and Arnoud Sonnenberg — 2011-12-01

The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule-actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.

The role of nesprins as multifunctional organizers in the nucleus and the cytoskeleton

Angelika A. Noegel and Sascha Neumann — 2011-12-01

Nesprins (nuclear envelope spectrin repeat proteins), also known as SYNE (synaptic nuclear envelope protein), MYNE (myocyte nuclear envelope protein), ENAPTIN and NUANCE, are proteins that are primarily components of the nuclear envelope. The nuclear envelope is a continuous membrane system composed of two lipid bilayers: an inner and an outer nuclear membrane. Nesprins are components of both nuclear membranes and reach into the nucleoplasm and the cytoplasm, where they undergo different interactions and have the potential to influence transcriptional processes and cytoskeletal activities.

Keeping the LINC: the importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function

Maria L. Lombardi and Jan Lammerding — 2011-12-01

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.

Evolvement of LEM proteins as chromatin tethers at the nuclear periphery

Andreas Brachner and Roland Foisner — 2011-12-01

The nuclear envelope in eukaryotic cells has important roles in chromatin organization. The inner nuclear membrane contains over 60 transmembrane proteins. LEM [LAP2 (lamina-associated polypeptide 2)/emerin/MAN1] domain-containing proteins of the inner nuclear membrane are involved in tethering chromatin to the nuclear envelope and affect gene expression. They contain a common structural, bihelical motif, the so-called LEM domain, which mediates binding to a conserved chromatin protein, BAF (barrier to autointegration factor). Interestingly, this domain is highly related to other bihelical motifs, termed HeH (helix-extension-helix) and SAP {SAF (scaffold attachment factor)/acinus/PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)]} motifs, which are directly linked to DNA. In the present paper, we summarize evidence that the LEM motif evolved from the HeH and SAP domains concomitantly with BAF. In addition, we discuss the potential evolution of HeH/SAP and LEM domain-containing proteins and their role in chromatin tethering and gene regulation from unicellular eukaryotes to mammals.

Nuclear envelope influences on cell-cycle progression

Vlastimil Srsen, Nadia Korfali and Eric C. Schirmer — 2011-12-01

The nuclear envelope is a complex double membrane system that serves as a dynamic interface between the nuclear and cytoplasmic compartments. Among its many roles is to provide an anchor for gene regulatory proteins on its nucleoplasmic surface and for the cytoskeleton on its cytoplasmic surface. Both sets of anchors are proteins called NETs (nuclear envelope transmembrane proteins), embedded respectively in the inner or outer nuclear membranes. Several lines of evidence indicate that the nuclear envelope contributes to cell-cycle regulation. These contributions come from both inner and outer nuclear membrane NETs and appear to operate through several distinct mechanisms ranging from sequestration of gene-regulatory proteins to activating kinase cascades.

Chromobility: the rapid movement of chromosomes in interphase nuclei

Joanna M. Bridger — 2011-12-01

There are an increasing number of studies reporting the movement of gene loci and whole chromosomes to new compartments within interphase nuclei. Some of the movements can be rapid, with relocation of parts of the genome within less than 15 min over a number of microns. Some of these studies have also revealed that the activity of motor proteins such as actin and myosin are responsible for these long-range movements of chromatin. Within the nuclear biology field, there remains some controversy over the presence of an active nuclear acto-myosin motor in interphase nuclei. However, both actin and myosin isoforms are localized to the nucleus, and there is a requirement for rapid and directed movements of genes and whole chromosomes and evidence for the involvement of motor proteins in this relocation. The presence of nuclear motors for chromatin movement is thus an important and timely debate to have.

LMNA-linked lipodystrophies: from altered fat distribution to cellular alterations

2011-12-01

Mutations in the LMNA gene, encoding the nuclear intermediate filaments the A-type lamins, result in a wide variety of diseases known as laminopathies. Some of them, such as familial partial lipodystrophy of Dunnigan and metabolic laminopathies, are characterized by lipodystrophic syndromes with altered fat distribution and severe metabolic alterations with insulin resistance and dyslipidaemia. Metabolic disturbances could be due either to the inability of adipose tissue to adequately store triacylglycerols or to other cellular alterations linked to A-type lamin mutations. Indeed, abnormal prelamin A accumulation and farnesylation, which are clearly involved in laminopathic premature aging syndromes, could play important roles in lipodystrophies. In addition, gene expression alterations, and signalling abnormalities affecting SREBP1 (sterol-regulatory-element-binding protein 1) and MAPK (mitogen-activated protein kinase) pathways, could participate in the pathophysiological mechanisms leading to LMNA (lamin A/C)-linked metabolic alterations and lipodystrophies. In the present review, we describe the clinical phenotype of LMNA-linked lipodystrophies and discuss the current physiological and biochemical hypotheses regarding the pathophysiology of these diseases.

Synthesis, transport and incorporation into the nuclear envelope of A-type lamins and inner nuclear membrane proteins

Jose M. González and Vicente Andrés — 2011-12-01

The mammalian NE (nuclear envelope), which separates the nucleus from the cytoplasm, is a complex structure composed of nuclear pore complexes, the outer and inner nuclear membranes, the perinuclear space and the nuclear lamina (A- and B-type lamins). The NE is completely disassembled and reassembled at each cell division. In the present paper, we review recent advances in the understanding of the mechanisms implicated in the transport of inner nuclear membrane and nuclear lamina proteins from the endoplasmic reticulum to the nucleus in interphase cells and mitosis, with special attention to A-type lamins.

DNA-damage accumulation and replicative arrest in Hutchinson-Gilford progeria syndrome

Phillip R. Musich and Yue Zou — 2011-12-01

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

Reproductive aging: insights from model organisms

Alice L. Ye and Needhi Bhalla — 2011-12-01

Aging was once thought to be the result of a general deterioration of tissues as opposed to their being under regulatory control. However, investigations in a number of model organisms have illustrated that aspects of aging are controlled by genetic mechanisms and are potentially manipulable, suggesting the possibility of treatment for age-related disorders. Reproductive decline is one aspect of aging. In model organisms and humans of both sexes, increasing age is associated with both a decline in the number of progeny and an increased incidence of defects. The cellular mechanisms of reproductive aging are not well understood, although a number of factors, both intrinsic and extrinsic to an organism's germline, may contribute to aging phenotypes. Recent work in a variety of organisms suggests that nuclear organization and nuclear envelope proteins may play a role in these processes.

In vitro pathological modelling using patient-specific induced pluripotent stem cells: the case of progeria

Xavier Nissan, Sophie Blondel and Marc Peschanski — 2011-12-01

Progeria, also known as HGPS (Hutchinson-Gilford progeria syndrome), is a rare fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (C1804T) of the gene encoding lamins A and C, LMNA, leading to the production of a truncated form of the protein called progerin. Owing to their unique potential to self-renew and to differentiate into any cell types of the organism, pluripotent stem cells offer a unique tool to study molecular and cellular mechanisms related to this global and systemic disease. Recent studies have exploited this potential by generating human induced pluripotent stem cells from HGPS patients' fibroblasts displaying several phenotypic defects characteristic of HGPS such as nuclear abnormalities, progerin expression, altered DNA-repair mechanisms and premature senescence. Altogether, these findings provide new insights on the use of pluripotent stem cells for pathological modelling and may open original therapeutic perspectives for diseases that lack pre-clinical in vitro human models, such as HGPS.

Defective DNA-damage repair induced by nuclear lamina dysfunction is a key mediator of smooth muscle cell aging

Derek T. Warren and Catherine M. Shanahan — 2011-12-01

Accumulation of DNA damage is a major driving force of normal cellular aging and has recently been demonstrated to hasten the development of vascular diseases such as atherosclerosis. VSMCs (vascular smooth muscle cells) are essential for vessel wall integrity and repair, and maintenance of their proliferative capacity is essential for vascular health. The signalling pathways that determine VSMC aging remain poorly defined; however, recent evidence implicates persistent DNA damage and the A-type nuclear lamins as key regulators of this process. In the present review, we discuss the importance of the nuclear lamina in the spatial organization of nuclear signalling events, including the DNA-damage response. In particular, we focus on the evidence suggesting that prelamin A accumulation interferes with nuclear spatial compartmentalization by disrupting chromatin organization and DNA-damage repair pathways to promote VSMC aging and senescence.

Microtubule-associated nuclear envelope proteins in interphase and mitosis

Ricardo A. Figueroa, Santhosh Gudise and Einar Hallberg — 2011-12-01

The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE (nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome relative to the NE is of vital importance for many cell functions, such as cell migration and division, and centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane domain associated with the mitotic spindle. The present review discusses structural and functional aspects of microtubule association with NE proteins and how this association may be maintained over the cell cycle.

UnLINCing the nuclear envelope: towards an understanding of the physiological significance of nuclear positioning

David Razafsky, Shulun Zang and Didier Hodzic — 2011-12-01

Appropriate tissue morphogenesis strictly requires the developmental regulation of different types of nuclear movements. LINC (linker of nucleoskeleton and cytoskeleton) complexes are macromolecular scaffolds that span the nuclear envelope and physically connect the nuclear interior to different cytoskeletal elements and molecular motors, thereby playing essential roles in nucleokinesis. Recent studies dedicated to the in vivo disruption of LINC complexes not only confirmed their widespread role in nuclear dynamics, but also led to a vigorous regain of interest in the physiological relevance of nuclear positioning within cells and syncitia. In the present paper, we review the results of LINC complex disruption in vivo across different organisms and the potential implications of observed phenotypes in human diseases.

The nuclear envelope and its involvement in cellular stress responses

Ashraf N. Malhas and David J. Vaux — 2011-12-01

The nuclear envelope is not only important for the structural integrity of the nucleus, but also involved in a number of cellular functions. It has been shown to be important for maintaining and controlling chromatin organization, sequestering transcription factors, replication, transcription and signalling. The nuclear envelope is thus important for development and differentiation, and some of its components are essential for cell viability. Among the many functions which are emerging for the nuclear envelope is its involvement in protecting the cell against different types of cellular stress. In the present paper, we review key findings which describe the roles of nuclear envelope components in responses to common types of stress conditions.

The metagenomic basis of anammox metabolism in Candidatus 'Brocadia fulgida'

2011-12-01

Anammox (anaerobic ammonium oxidation) coupled to nitrite reduction is an important step in the nitrogen cycle and has been recognized as an important sink for fixed nitrogen in the ocean. Still little is known about the genomic blueprint of different anammox species. In the present article, we discuss the important genes of anammox metabolism in Candidatus 'Brocadia fulgida' that were retrieved via a metagenomic approach.

The ultrastructure of the compartmentalized anaerobic ammonium-oxidizing bacteria is linked to their energy metabolism

Sarah Neumann, Mike S.M. Jetten and Laura van Niftrik — 2011-12-01

The most striking example of a complex prokaryotic intracytoplasmic organization can be found in the members of the phylum Planctomycetes. Among them are the anammox (anaerobic ammonium-oxidizing) bacteria, which possess a unique cell compartment with an unprecedented function in bacteria: the anammoxosome is a prokaryotic cell organelle evolved for energy metabolism. It is an independent entity, which is enclosed by a contiguous membrane. Several lines of evidence indicate its importance in the anammox reaction and the unusual subcellular organization may well be essential for the lifestyle of anammox bacteria. The present review summarizes our knowledge about the ultrastructure of anammox cells and the connection between the anammoxosome and the energy metabolism of the cell. In the future, much more research will be necessary to validate the current models and to answer questions on the functional cell biology of anammox bacteria.

Anaerobic ammonium oxidation (anammox) in different natural ecosystems

Bao‑lan Hu, Li‑dong Shen, Xiang‑yang Xu and Ping Zheng — 2011-12-01

Anammox (anaerobic ammonium oxidation), which is a reaction that oxidizes ammonium to dinitrogen gas using nitrite as the electron acceptor under anoxic conditions, was an important discovery in the nitrogen cycle. The reaction is mediated by a specialized group of planctomycete-like bacteria that were first discovered in man-made ecosystems. Subsequently, many studies have reported on the ubiquitous distribution of anammox bacteria in various natural habitats, including anoxic marine sediments and water columns, freshwater sediments and water columns, terrestrial ecosystems and some special ecosystems, such as petroleum reservoirs. Previous studies have estimated that the anammox process is responsible for 50% of the marine nitrogen loss. Recently, the anammox process was reported to account for 9-40% and 4-37% of the nitrogen loss in inland lakes and agricultural soils respectively. These findings indicate the great potential for the anammox process to occur in freshwater and terrestrial ecosystems. The distribution of different anammox bacteria and their contribution to nitrogen loss have been described in different natural habitats, demonstrating that the anammox process is strongly influenced by the local environmental conditions. The present mini-review summarizes the current knowledge of the ecological distribution of anammox bacteria, their contribution to nitrogen loss in various natural ecosystems and the effects of major influential factors on the anammox process.

Anammox bacteria in different compartments of recirculating aquaculture systems

2011-12-01

Strict environmental restrictions force the aquaculture industry to guarantee optimal water quality for fish production in a sustainable manner. The implementation of anammox (anaerobic ammonium oxidation) in biofilters would result in the conversion of both ammonium and nitrite (both toxic to aquatic animals) into harmless dinitrogen gas. Both marine and freshwater aquaculture systems contain populations of anammox bacteria. These bacteria are also present in the faeces of freshwater and marine fish. Interestingly, a new planctomycete species appears to be present in these recirculation systems too. Further exploitation of anammox bacteria in different compartments of aquaculture systems can lead to a more environmentally friendly aquaculture practice.

Combined anaerobic ammonium and methane oxidation for nitrogen and methane removal

2011-12-01

Anammox (anaerobic ammonium oxidation) is an environment-friendly and cost-efficient nitrogen-removal process currently applied to high-ammonium-loaded wastewaters such as anaerobic digester effluents. In these wastewaters, dissolved methane is also present and should be removed to prevent greenhouse gas emissions into the environment. Potentially, another recently discovered microbial pathway, n-damo (nitrite-dependent anaerobic methane oxidation) could be used for this purpose. In the present paper, we explore the feasibility of simultaneously removing methane and ammonium anaerobically, starting with granules from a full-scale anammox bioreactor. We describe the development of a co-culture of anammox and n-damo bacteria using a medium containing methane, ammonium and nitrite. The results are discussed in the context of other recent studies on the application of anaerobic methane- and ammonia-oxidizing bacteria for wastewater treatment.

Nitrifying and denitrifying pathways of methanotrophic bacteria

Lisa Y. Stein and Martin G. Klotz — 2011-12-01

Nitrous oxide, a potent greenhouse gas and ozone-depleting molecule, continues to accumulate in the atmosphere as a product of anthropogenic activities and land-use change. Nitrogen oxides are intermediates of nitrification and denitrification and are released as terminal products under conditions such as high nitrogen load and low oxygen tension among other factors. The rapid completion and public availability of microbial genome sequences has revealed a high level of enzymatic redundancy in pathways terminating in nitrogen oxide metabolites, with few enzymes involved in returning nitrogen oxides to dinitrogen. The aerobic methanotrophic bacteria are particularly useful for discovering and analysing diverse mechanisms for nitrogen oxide production, as these microbes both nitrify (oxidize ammonia to nitrite) and denitrify (reduce nitrate/nitrite to nitrous oxide via nitric oxide), and yet do not rely on these pathways for growth. The fact that methanotrophs have a rich inventory for nitrogen oxide metabolism is, in part, a consequence of their evolutionary relatedness to ammonia-oxidizing bacteria. Furthermore, the ability of individual methanotrophic taxa to resist toxic intermediates of nitrogen metabolism affects the relative abundance of nitrogen oxides released into the environment, the composition of their community, and the balance between nitrogen and methane cycling.

Nitrous oxide production by lithotrophic ammonia-oxidizing bacteria and implications for engineered nitrogen-removal systems

Kartik Chandran, Lisa Y. Stein, Martin G. Klotz and Mark C.M. van Loosdrecht — 2011-12-01

Chemolithoautotrophic AOB (ammonia-oxidizing bacteria) form a crucial component in microbial nitrogen cycling in both natural and engineered systems. Under specific conditions, including transitions from anoxic to oxic conditions and/or excessive ammonia loading, and the presence of high nitrite (NO₂⁻) concentrations, these bacteria are also documented to produce nitric oxide (NO) and nitrous oxide (N₂O) gases. Essentially, ammonia oxidation in the presence of non-limiting substrate concentrations (ammonia and O₂) is associated with N₂O production. An exceptional scenario that leads to such conditions is the periodical switch between anoxic and oxic conditions, which is rather common in engineered nitrogen-removal systems. In particular, the recovery from, rather than imposition of, anoxic conditions has been demonstrated to result in N₂O production. However, applied engineering perspectives, so far, have largely ignored the contribution of nitrification to N₂O emissions in greenhouse gas inventories from wastewater-treatment plants. Recent field-scale measurements have revealed that nitrification-related N₂O emissions are generally far higher than emissions assigned to heterotrophic denitrification. In the present paper, the metabolic pathways, which could potentially contribute to NO and N₂O production by AOB have been conceptually reconstructed under conditions especially relevant to engineered nitrogen-removal systems. Taken together, the reconstructed pathways, field- and laboratory-scale results suggest that engineering designs that achieve low effluent aqueous nitrogen concentrations also minimize gaseous nitrogen emissions.

Bacterial nitrate assimilation: gene distribution and regulation

2011-12-01

In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.

A haloarchaeal ferredoxin electron donor that plays an essential role in nitrate assimilation

2011-12-01

In the absence of ammonium, many organisms, including the halophilic archaeon Haloferax volcanii DS2 (DM3757), may assimilate inorganic nitrogen from nitrate or nitrite, using a ferredoxin-dependent assimilatory NO₃⁻/NO₂⁻ reductase pathway. The small acidic ferredoxin Hv-Fd plays an essential role in the electron transfer cascade required for assimilatory nitrate and nitrite reduction by the cytoplasmic NarB- and NirA-type reductases respectively. UV-visible absorbance and EPR spectroscopic characterization of purified Hv-Fd demonstrate that this protein binds a single [2Fe-2S] cluster, and potentiometric titration reveals that the cluster shares similar redox properties with those present in plant-type ferredoxins.

Metabolic adaptation of Pseudomonas pseudoalcaligenes CECT5344 to cyanide: role of malate-quinone oxidoreductases, aconitase and fumarase isoenzymes

2011-12-01

In general, the biodegradation of a toxic compound by a micro-organism requires the concurrence of, at least, two features in the biological system: first, the capability of the micro-organism to metabolize the toxic compound, and secondly, the capacity to resist its toxic effect. Pseudomonas pseudoalcaligenes CECT5344 is a bacterium used in the biodegradation of cyanide because it is capable to use it as a nitrogen source. The present review is mainly focused on the putative role of iron-containing enzymes of the tricarboxylic acid cycle in cyanide resistance by P. pseudoalcaligenes CECT5344.

Role of Fur on cyanide tolerance of Pseudomonas pseudoalcaligenes CECT5344

2011-12-01

Pseudomonas pseudoalcaligenes CECT5344 can be used in cyanide bioremediation processes because it grows at pH 9.5 using 2.0 mM cyanide at the sole nitrogen source. Cyanide strongly binds to metals creating iron-deprivation conditions. The bacterium responds to the presence of cyanide by inducing several processes such as siderophore synthesis for iron capture, cyanide-insensitive respiration system and defence mechanisms against oxidative stress. Since high concentrations of cyanide cause iron deficiency and because iron is an essential nutrient, bacterial growth in the presence of cyanide requires an efficient iron uptake. Fur is a global transcription factor that regulates a diversity of biological processes such as iron homoeostasis, TCA (tricarboxylic acid) cycle metabolism and oxidative stress response. Fur's regulation of iron uptake and storage genes should play a significant role in the lives of these bacteria. In the present review, current knowledge of Fur is summarized.

Dissimilatory sulfur cycling in oxygen minimum zones: an emerging metagenomics perspective

Frank J. Stewart — 2011-12-01

Biological diversity in marine OMZs (oxygen minimum zones) is dominated by a complex community of bacteria and archaea whose anaerobic metabolisms mediate key steps in global nitrogen and carbon cycles. Molecular and physiological studies now confirm that OMZs also support diverse micro-organisms capable of utilizing inorganic sulfur compounds for energy metabolism. The present review focuses specifically on recent metagenomic data that have helped to identify the molecular basis for autotrophic sulfur oxidation with nitrate in the OMZ water column, as well as a cryptic role for heterotrophic sulfate reduction. Interpreted alongside marker gene surveys and process rate measurements, these data suggest an active sulfur cycle with potentially substantial roles in organic carbon input and mineralization and critical links to the OMZ nitrogen cycle. Furthermore, these studies have created a framework for comparing the genomic diversity and ecology of pelagic sulfur-metabolizing communities from diverse low-oxygen regions.

Physiological function and catalytic versatility of bacterial multihaem cytochromes c involved in nitrogen and sulfur cycling

Jörg Simon, Melanie Kern, Bianca Hermann, Oliver Einsle and Julea N. Butt — 2011-12-01

Bacterial MCCs (multihaem cytochromes c) represent widespread respiratory electron-transfer proteins. In addition, some of them convert substrates such as nitrite, hydroxylamine, nitric oxide, hydrazine, sulfite, thiosulfate or hydrogen peroxide. In many cases, only a single function is assigned to a specific MCC in database entries despite the fact that an MCC may accept various substrates, thus making it a multifunctional catalyst that can play diverse physiological roles in bacterial respiration, detoxification and stress defence mechanisms. The present article briefly reviews the structure, function and biogenesis of selected MCCs that catalyse key reactions in the biogeochemical nitrogen and sulfur cycles.

Characterization of the active site and calcium binding in cytochrome c nitrite reductases

2011-12-01

The decahaem homodimeric cytochrome c nitrite reductase (NrfA) is expressed within the periplasm of a wide range of Gamma-, Delta- and Epsilon-proteobacteria and is responsible for the six-electron reduction of nitrite to ammonia. This allows nitrite to be used as a terminal electron acceptor, facilitating anaerobic respiration while allowing nitrogen to remain in a biologically available form. NrfA has also been reported to reduce nitric oxide (a reaction intermediate) and sulfite to ammonia and sulfide respectively, suggesting a potential secondary role as a detoxification enzyme. The protein sequences and crystal structures of NrfA from different bacteria and the closely related octahaem nitrite reductase from Thioalkalivibrio nitratireducens (TvNir) reveal that these enzymes are homologous. The NrfA proteins contain five covalently attached haem groups, four of which are bis-histidine-co-ordinated, with the proximal histidine being provided by the highly conserved CXXCH motif. These haems are responsible for intraprotein electron transfer. The remaining haem is the site for nitrite reduction, which is ligated by a novel lysine residue provided by a CXXCK haem-binding motif. The TvNir nitrite reductase has five haems that are structurally similar to those of NrfA and three extra bis-histidine-coordinated haems that precede the NrfA conserved region. The present review compares the protein sequences and structures of NrfA and TvNir and discusses the subtle differences related to active-site architecture and Ca²⁺ binding that may have an impact on substrate reduction.

The production and detoxification of a potent cytotoxin, nitric oxide, by pathogenic enteric bacteria

Anke Arkenberg, Sebastian Runkel, David J. Richardson and Gary Rowley — 2011-12-01

The nitrogen cycle is based on several redox reactions that are mainly accomplished by prokaryotic organisms, some archaea and a few eukaryotes, which use these reactions for assimilatory, dissimilatory or respiratory purposes. One group is the Enterobacteriaceae family of Gammaproteobacteria, which have their natural habitats in soil, marine environments or the intestines of humans and other warm-blooded animals. Some of the genera are pathogenic and usually associated with intestinal infections. Our body possesses several physical and chemical defence mechanisms to prevent pathogenic enteric bacteria from invading the gastrointestinal tract. One response of the innate immune system is to activate macrophages, which produce the potent cytotoxin nitric oxide (NO). However, some pathogens have evolved the ability to detoxify NO to less toxic compounds, such as the neuropharmacological agent and greenhouse gas nitrous oxide (N₂O), which enables them to overcome the host's attack. The same mechanisms may be used by bacteria producing NO endogenously as a by-product of anaerobic nitrate respiration. In the present review, we provide a brief introduction into the NO detoxification mechanisms of two members of the Enterobacteriaceae family: Escherichia coli and Salmonella enterica serovar Typhimurium. These are discussed as comparative non-pathogenic and pathogenic model systems in order to investigate the importance of detoxifying NO and producing N₂O for the pathogenicity of enteric bacteria.

The nitric oxide response in plant-associated endosymbiotic bacteria

2011-12-01

Nitric oxide (NO) is a gaseous signalling molecule which becomes very toxic due to its ability to react with multiple cellular targets in biological systems. Bacterial cells protect against NO through the expression of enzymes that detoxify this molecule by oxidizing it to nitrate or reducing it to nitrous oxide or ammonia. These enzymes are haemoglobins, c-type nitric oxide reductase, flavorubredoxins and the cytochrome c respiratory nitrite reductase. Expression of the genes encoding these enzymes is controlled by NO-sensitive regulatory proteins. The production of NO in rhizobia-legume symbiosis has been demonstrated recently. In functioning nodules, NO acts as a potent inhibitor of nitrogenase enzymes. These observations have led to the question of how rhizobia overcome the toxicity of NO. Several studies on the NO response have been undertaken in two non-dentrifying rhizobial species, Sinorhizobium meliloti and Rhizobium etli, and in a denitrifying species, Bradyrhizobium japonicum. In the present mini-review, current knowledge of the NO response in those legume-associated endosymbiotic bacteria is summarized.

Denitrification in Sinorhizobium meliloti

2011-12-01

Denitrification is the complete reduction of nitrate or nitrite to N₂, via the intermediates nitric oxide (NO) and nitrous oxide (N₂O), and is coupled to energy conservation and growth under O₂-limiting conditions. In Bradyrhizobium japonicum, this process occurs through the action of the napEDABC, nirK, norCBQD and nosRZDFYLX gene products. DNA sequences showing homology with nap, nirK, nor and nos genes have been found in the genome of the symbiotic plasmid pSymA of Sinorhizobium meliloti strain 1021. Whole-genome transcriptomic analyses have demonstrated that S. meliloti denitrification genes are induced under micro-oxic conditions. Furthermore, S. meliloti has also been shown to possess denitrifying activities in both free-living and symbiotic forms. Despite possessing and expressing the complete set of denitrification genes, S. meliloti is considered a partial denitrifier since it does not grow under anaerobic conditions with nitrate or nitrite as terminal electron acceptors. In the present paper, we show that, under micro-oxic conditions, S. meliloti is able to grow by using nitrate or nitrite as respiratory substrates, which indicates that, in contrast with anaerobic denitrifiers, O₂ is necessary for denitrification by S. meliloti. Current knowledge of the regulation of S. meliloti denitrification genes is also included.

A snapshot of a pathogenic bacterium mid-evolution: Neisseria meningitidis is becoming a nitric oxide-tolerant aerobe

James W. Moir — 2011-12-01

Members of the Neisseria genus typically display the ability to carry out denitrification of nitrite to nitrous oxide as an alternative to oxygen respiration when oxygen is depleted. The key enzymes nitrite and nitric oxide reductase are found across the Neisseria genus. Within Neisseria meningitidis, however, a number of research groups have found that a significant proportion of strains lack a functional nitrite reductase. It appears that N. meningitidis is on an evolutionary trajectory towards loss of the capacity to reduce nitrite. In the present paper, I propose that N. meningitidis is evolving to become a nitric oxide-tolerant aerobe in order to occupy an oxygen-rich niche close to host tissue (and hence oxygen perfusion). Other features of the genomic and functional specialization of N. meningitidis, such as possession of a polysaccharide capsule and various acquired reactive oxygen species-resistance mechanisms, support this proposition. The importance of oxygen availability more generally is discussed with reference to recent findings with other mucosal pathogens.

Tied down: tethering redox proteins to the outer membrane in Neisseria and other genera

Xi Li, Steven Parker, Manu Deeudom and James W. Moir — 2011-12-01

Typically, the redox proteins of respiratory chains in Gram-negative bacteria are localized in the cytoplasmic membrane or in the periplasm. An alternative arrangement appears to be widespread within the betaproteobacterial genus Neisseria, wherein several redox proteins are covalently associated with the outer membrane. In the present paper, we discuss the structural properties of these outer membrane redox proteins and the functional consequences of this attachment. Several tethered outer membrane redox proteins of Neisseria contain a weakly conserved repeated structure between the covalent tether and the redox protein globular domain that should enable the redox cofactor-containing domain to extend from the outer membrane, across the periplasm and towards the inner membrane. It is argued that the constraints imposed on the movement and orientation of the globular domains by these tethers favours the formation of electron-transfer complexes for entropic reasons. The attachment to the outer membrane may also affect the exposure of the host to redox proteins with a moonlighting function in the host-microbe interaction, thus affecting the host response to Neisseria infection. We identify putative outer membrane redox proteins from a number of other bacterial genera outside Neisseria, and suggest that this organizational arrangement may be more common than previously recognized.

Vacuole-mitochondrial cross-talk during apoptosis in yeast: a model for understanding lysosome-mitochondria-mediated apoptosis in mammals

2011-12-01