TZAP‐ing telomeres down to size

The phenomenon of gradual telomere shortening has become a paradigm for how we understand the biology of aging and cancer. Cell proliferation is accompanied by cumulative telomere loss, and the aged cell either senesces, dies or transforms toward cancer. This transformation requires the activation of telomere elongation mechanisms in order to restore telomere length such that cell death or senescence programs are not induced. Most of the time, this occurs through telomerase reactivation. In other rare cases, the Alternative lengthening of telomeres (ALT) pathway hijacks DNA recombination‐associated mechanisms to hyperextend telomeres, often to more than 50 kb. Why telomere length is restricted and what sets their maximal length has been a long‐standing puzzle in cell biology. Two recent studies published in this issue of EMBO Reports [1] and recently in Science [2] sought to address this important question. Both built on omics approaches that identified ZBTB48 as a potential telomere‐associated protein and reveal it to be a critical regulator of telomere length homeostasis by the telomere trimming mechanism. These discoveries provide fundamental insights for our understanding of telomere trimming and how it impacts telomere integrity in stem and cancer cells.     

Effects of NIX‐mediated mitophagy on ox‐LDL‐induced macrophage pyroptosis in atherosclerosis

Pyroptosis is a form of cell death that is uniquely dependent on caspase‐1. Pyroptosis involved in oxidized low‐density lipoprotein (ox‐LDL)‐induced human macrophage death through the promotion of caspase‐1 activation is important for the formation of unstable plaques in atherosclerosis. The mitochondrial outer membrane protein NIX directly interacts with microtubule‐associated protein 1 light chain 3 (LC3). Although we previously showed that NIX‐mediated mitochondrial autophagy is involved in the clearance of damaged mitochondria, how NIX contributes to ox‐LDL‐induced macrophage pyroptosis remains unknown. Here, immunoperoxidase staining Nix expression decreased in human atherosclerosis. When we silenced NIX expression in murine macrophage cell, active caspase‐1, and mature interleukin‐1β expression levels were increased and LC3 was reduced. In addition, LDH release and acridine orange and ethidium bromide staining indicated that damage to macrophage cell membranes induced by ox‐LDL was substantially worse. Moreover, intracellular reactive oxygen species and NLRP3 inflammasome levels increased. Taken together, these results demonstrated that NIX inhibits ox‐LDL‐induced macrophage pyroptosis via autophagy in atherosclerosis.     

Consequences of electroshock‐induced narcosis in fish muscle: from mitochondria to swim performance

Adult zebrafish Danio rerio were exposed to an electric shock of 3 V and 1A for 5 s delivered by field backpack electrofishing gear, to induce a taxis followed by a narcosis. The effect of such electric shock was investigated on both the individual performances (swimming capacities and costs of transport) and at cellular and mitochondrial levels (oxygen consumption and oxidative balance). The observed survival rate was very high (96·8%) independent of swimming speed (up to 10 body length s^?1). The results showed no effect of the treatment on the metabolism and cost of transport of the fish. Nor did the electroshock trigger any changes on muscular oxidative balance and bioenergetics even if red muscle fibres were more oxidative than white muscle. Phosphorylating respiration rates rose between (mean 1 s.e. ) 11·16 ± 1·36 pmol O₂ s^?1 mg^?1 and 15·63 ± 1·60 pmol O₂ s^?1 mg^?1 for red muscle fibres whereas phosphorylating respiration rates only reached 8·73 ± 1·27 pmol O₂ s^?1 mg^?1 in white muscle. Such an absence of detectable physiological consequences after electro‐induced narcosis both at organismal and cellular scales indicate that this capture method has no apparent negative post‐shock performance under the conditions of this study.     

The pH sensitivity of histidine‐contain‐ ing lytic peptides

Many bioactive peptides are featured by their unique amino acid compositions such as argine/lysine‐rich peptides. However, histidine‐rich bioactive peptides are hardly found. In this study, histidine‐containing peptides were constructed by selectively replacing the corresponded lysine residues in a lytic peptide LL‐1 with histidines. Interestingly, all resulting peptides demonstrated pH‐dependent activities. The cell lysis activities of these peptides could be increased up to four times as the solution pHs dropped from pH = 7.4 to pH = 5.5. The pH sensitivity of a histidine‐containing peptide was determined by histidine substitution numbers. Peptide derivatives with more histidines were associated with increased pH sensitivity. Results showed that not the secondary structures but pH‐affected cell affinity changes were responsible for the pH‐dependent activities of histidine‐containing peptides. The histidine substitution approach demonstrated here may present a general strategy to construct bioactive peptides with desired pH sensitivity for various applications. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Movement of labelled metabolites from mitochondria to plastids during development

Summary The exchange of metabolites from isolated mitochondria of Avena sativa L. to isolated etioplasts or 1–24 h etio-chloroplasts, from the same laminae, has been investigated. The results confirm the synchronised changes in the permeabilities of the inner membranes of each during plastid development. They also indicated the possibility of directed transport of certain metabolites from mitochondria to plastids especially during the early stages of chloroplast maturation. Over 80% of labelled succinate and oxaloacetate previously associated with mitochondria was found to be associated with 1 and 2 h etio-chloroplasts after in vitro incubation, with their respective mitochondria, for only 1 min at 0°C. Other metabolites showed variable but lower rates of transfer and that of aminolevulinic acid was at a much reduced level throughout development.Abbreviation ALA amino-levulinic acid     

Tendon‐to‐bone attachment: From development to maturity

The attachment between tendon and bone occurs across a complex transitional tissue that minimizes stress concentrations and allows for load transfer between muscles and skeleton. This unique tissue cannot be reconstructed following injury, leading to high incidence of recurrent failure and stressing the need for new clinical approaches. This review describes the current understanding of the development and function of the attachment site between tendon and bone. The embryonic attachment unit, namely, the tip of the tendon and the bone eminence into which it is inserted, was recently shown to develop modularly from a unique population of Sox9‐ and Scx‐positive cells, which are distinct from tendon fibroblasts and chondrocytes. The fate and differentiation of these cells is regulated by transforming growth factor beta and bone morphogenetic protein signaling, respectively. Muscle loads are then necessary for the tissue to mature and mineralize. Mineralization of the attachment unit, which occurs postnatally at most sites, is largely controlled by an Indian hedgehog/parathyroid hormone‐related protein feedback loop. A number of fundamental questions regarding the development of this remarkable attachment system require further study. These relate to the signaling mechanism that facilitates the formation of an interface with a gradient of cellular and extracellular phenotypes, as well as to the interactions between tendon and bone at the point of attachment. Birth Defects Research (Part C) 102:101–112, 2014. © 2014 Wiley Periodicals, Inc.     

PTEN: from pathology to biology

The PTEN tumour suppressor gene is mutated frequently in many malignancies and its importance in the development of cancer is probably underestimated. As the primary phosphatase of phosphatidylinositol (3,4,5)-trisphosphate, PTEN has a central role in reigning in the phosphoinositide 3-kinase (PI 3-kinase) network to control cellular homeostasis. Cells that lack PTEN are unable to regulate the PtdIns 3-kinase programme, which stimulates a variety of cellular phenotypes that favour oncogenesis. As well as the well-known role as tumour suppressor, recent studies show that PTEN is involved in the regulation of several basic cellular functions, such as cell migration, cell size, contractility of cardiac myocytes and chemotaxis. Here, we review the roles of PTEN in normal cellular functions and disease development.     

Spontaneous development of Alzheimer's disease‐associated brain pathology in a Shugoshin‐1 mouse cohesinopathy model

Spontaneous late‐onset Alzheimer's disease (LOAD) accounts for more than 95% of all human AD. As mice do not normally develop AD and as understanding on molecular processes leading to spontaneous LOAD has been insufficient to successfully model LOAD in mouse, no mouse model for LOAD has been available. Existing mouse AD models are all early‐onset AD (EOAD) models that rely on forcible expression of AD‐associated protein(s), which may not recapitulate prerequisites for spontaneous LOAD. This limitation in AD modeling may contribute to the high failure rate of AD drugs in clinical trials. In this study, we hypothesized that genomic instability facilitates development of LOAD and tested two genomic instability mice models in the brain pathology at the old age. Shugoshin‐1 (Sgo1) haploinsufficient (?) mice, a model of chromosome instability (CIN) with chromosomal and centrosomal cohesinopathy, spontaneously exhibited a major feature of AD pathology; amyloid beta accumulation that colocalized with phosphorylated Tau, beta‐secretase 1 (BACE), and mitotic marker phospho‐Histone H3 (p‐H3) in the brain. Another CIN model, spindle checkpoint‐defective BubR1^?/+ haploinsufficient mice, did not exhibit the pathology at the same age, suggesting the prolonged mitosis‐origin of the AD pathology. RNA‐seq identified ten differentially expressed genes, among which seven genes have indicated association with AD pathology or neuronal functions (e.g., ARC, EBF3). Thus, the model represents a novel model that recapitulates spontaneous LOAD pathology in mouse. The Sgo1^?/+ mouse may serve as a novel tool for investigating mechanisms of spontaneous progression of LOAD pathology, for early diagnosis markers, and for drug development.     

A further case of Dop‐ing in bacterial pupylation

Two recent studies, one in this issue of EMBO reports and one in Molecular Cell, identify Dop as a depupylase, ascribing a novel function to Dop and providing further evidence for the functional similarity of the prokaryotic Pup-modification system and the eukaryotic ubiquitin system.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.119Protein homeostasis is fundamental to the function of all cellular systems. In eukaryotes, the ubiquitin–proteasome pathway mediates regulated protein degradation. Intensive studies of the eukaryotic proteasome over the past decades have unravelled the complexity of this multi-subunit, ATP-dependent protease, and proteasome inhibitors are now established anticancer drugs (Finley, 2009). Prokaryotes use ATP-dependent proteases—such as Lon, ClpP and FtsH—for protein degradation. In addition, some bacteria in the class of Actinomycetes have acquired a proteasome which shares sequence and structural homology with its eukaryotic counterpart (Darwin, 2009). The function of the prokaryotic proteasome and its implication in pathogenesis is the subject of ongoing research. In Mycobacterium tuberculosis, proteasome activity is essential for the pathogen to persist in macrophages of the lung epithelium and could therefore be a target for antimicrobial treatment (Darwin, 2009).Labelling substrates for proteasomal degradation is well understood in eukaryotes, in which ubiquitin is attached to proteins that are subsequently recognized by proteasomal subunits and degraded (Finley, 2009). A similar tagging system has recently been identified in M. tuberculosis, in which the prokaryotic ubiquitin-like protein (Pup) serves as a ubiquitin analogue (Pearce et al, 2008). Subsequent proteome-wide studies have identified hundreds of Pup-tagged substrates in different mycobacteria, defining the ‘pupylome'' (Festa et al, 2010; Poulsen et al, 2010). Pupylated proteins are recognized by the proteasome-associated ATPase Mpa, that unfolds proteins before they are degraded in the proteolytic core (Darwin, 2009).Ubiquitination is reversed by specific deubiquitinases, but whether pupylation is also reversible was previously unknown. Two studies by Darwin and colleagues and—in this issue of EMBO reports—by Weber-Ban and colleagues have now demonstrated that Pup is removed from substrates when incubated with mycobacterial lysates (Burns et al, 2010; Imkamp et al, 2010). This suggests the presence of one or more ‘depupylases'', and indicates that pupylation is a complex and versatile process, much like ubiquitination.Pup and ubiquitin conjugation are mechanistically unrelated; ubiquitin is ligated by its carboxy-terminal glycine residue to lysine residues of target proteins by an enzymatic cascade, comprising E1, E2 and E3 enzymes (Dye & Schulman, 2007). By contrast, the pupylation machinery seems to be simpler; a single ligating enzyme, proteasome accessory factor A (PafA), mediates isopeptide bond formation between the C-terminal glutamic acid side-chain carboxyl group of Pup and a substrate lysine residue (Sutter et al, 2010).Only about half of the Pup-containing bacteria encode a glutamic acid residue at the C-terminus (Striebel et al, 2009). In the remaining species, including M. tuberculosis, the Pup gene encodes a C-terminal glutamine, which requires deamidation to glutamic acid before conjugation to substrates can occur. This activating deamidation step is carried out by the deamidase of Pup (Dop; Striebel et al, 2009). Curiously, the dop gene is conserved in all Pup-containing bacterial species (with the exception of Plesiocystis pacifica), including those in which initial deamidation is unnecessary.Imkamp et al and Burns et al now identify Dop as a depupylase in the Pup-modification pathway. Hydrolysis of Pup from model substrates in vitro is abolished in a dop-deficient bacterial lysate, or in lysate expressing a mutant form of dop, but can be restored by complementation with dop. Dop is able to depupylate many proteins when tested against the pupylome, suggesting a broad substrate spectrum. By contrast, without Dop the pupylome is unchanged over time, indicating that Dop might be the main depupylase in Mycobacteria. Purified Dop from M. tuberculosis shows depupylase activity against model substrates. Finally, Imkamp et al analyse a Dop homologue from Corynebacterium glutamicum that encodes Pup^Glu and hence does not depend on deamidation. This Dop homologue is expressed recombinantly and purified from Escherichia coli—which does not harbour the Pup-proteasome system—and shown to be an active depupylase in vitro.Both groups then investigated the functional relationship between Pup/Dop and the proteasomal ATPase Mpa. Burns et al found that Mpa is required in vivo for depupylation of a proteasome substrate. Imkamp et al found that Mpa significantly increases depupylation activity in vitro. The mechanism for this remains unclear, but full-length Pup seems to be essential for Mpa-mediated activation, as depupylation is not enhanced with an amino-terminally truncated Pup. Previous work has indicated that the N-terminus of Pup is required to initiate substrate unfolding (Striebel et al, 2010), and Imkamp et al speculated that unfolding makes the isopeptide bond more accessible for interaction with Dop. Evidence for this comes from the observation that Dop can cleave a peptide substrate with an accessible isopeptide bond at the same rate in the presence or absence of Mpa. It is intriguing that Dop co-purifies with the pupylome (Burns et al, 2010), this suggests that Dop has significant affinity but low activity for pupylated substrates. This might, however, prime the system for depupylation after Mpa interaction.Corynebacteria do not have a proteasome, but maintain the pupylation machinery comprising Pup, PafA, Dop and the proteasomal ATPase ARC (a homologue of Mpa). Here, the fate of Pup-tagged proteins cannot be proteasomal degradation, although substrate unfolding by ARC could initiate degradation by other proteases. However, pupylation in proteasome-deficient bacteria might suggest additional non-degradative functions for pupylation.Both studies demonstrate that Dop acts as a depupylase in Pup-containing bacteria, in addition to the previously reported deamidation role of Dop in mycobacteria. In fact, the chemical reactions underlying depupylation and deamidation are mechanistically similar. The key functional question that remains is whether Dop protects substrates from proteasomal degradation. Alternative explanations are that Dop acts in conjunction with Mpa or the proteasome to recycle Pup, or that it reverses non-degradative roles of pupylation (Fig 1).Open in a separate windowFigure 1Emerging roles for Dop. (A) The pupylation system. (1) Dop functions as a deamidase, converting Pup^Gln to Pup^Glu. PafA ligates Pup^Glu to substrates, which are targeted to Mpa and the proteasome and are degraded. (2) Dop can reverse pupylation on substrates and might rescue substrates from degradation. (B) Dop might act to recycle Pup, either (3a) at the Mpa/proteasome level or (3b) by binding to pupylated substrates, where Mpa-mediated substrate unfolding activates Dop. (C) (4) The existence of Dop in proteasome deficient bacteria might indicate that Dop antagonizes non-degradational roles for Pup. Dop, deamidase of Pup; Mpa, Mycobacterium proteasome-associated ATPase; PafA, proteasome accessory factor A; Pup, prokaryotic ubiquitin-like protein.So far, nothing is known about the regulation of Dop. It will be interesting to analyse expression profiles to determine whether Dop is regulated independently of other proteins in this system. Other open questions remain about the existence of co-factors and binding partners, and the organization of the Pup–Dop–Mpa network. Structural studies of the Dop enzyme will hopefully increase our understanding of its roles in depupylation.In conclusion, Dop in the pupylation system has the potential to combine all known functions of deubiquitinases in the ubiquitin system: processing of precursors, rescuing substrates from degradation, recycling the modifier and reversing potential non-degradative roles of pupylation. The identification of the first depupylase opens an exciting new research field to unravel the functional consequences of depupylation.     

Mouse lines with photo‐activatable mitochondria to study mitochondrial dynamics

Many pathological states involve dysregulation of mitochondrial fusion, fission, or transport. These dynamic events are usually studied in cells lines because of the challenges in tracking mitochondria in tissues. To investigate mitochondrial dynamics in tissues and disease models, we generated two mouse lines withphoto‐activatable mitochondria (PhAM). In the PhAM ^floxed line, a mitochondrially localized version of the photo‐convertible fluorescent protein Dendra2 (mito‐Dendra2) is targeted to the ubiquitously expressed Rosa26 locus, along with an upstream loxP‐flanked termination signal. Expression of Cre in PhAM ^floxed cells results in bright mito‐Dendra2 fluorescence without adverse effects on mitochondrial morphology. When crossed with Cre drivers, the PhAM ^floxed line expresses mito‐Dendra2 in specific cell types, allowing mitochondria to be tracked even in tissues that have high cell density. In a second line (PhAM ^excised), the expression of mito‐Dendra2 is ubiquitous, allowing mitochondria to be analyzed in a wide range of live and fixed tissues. By using photo‐conversion techniques, we directly measured mitochondrial fusion events in cultured cells as well as tissues such as skeletal muscle. These mouse lines facilitate analysis of mitochondrial dynamics in a wide spectrum of primary cells and tissues, and can be used to examine mitochondria in developmental transitions and disease states. © genesis 1–11, 2012. © 2012 Wiley Periodicals, Inc.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.     

AMP‐activated protein kinase mediates activity‐dependent axon branching by recruiting mitochondria to axon

During development, axons are guided to their target areas and provide local branching. Spatiotemporal regulation of axon branching is crucial for the establishment of functional connections between appropriate pre‐ and postsynaptic neurons. Common understanding has been that neuronal activity contributes to the proper axon branching; however, intracellular mechanisms that underlie activity‐dependent axon branching remain elusive. Here, we show, using primary cultures of the dentate granule cells, that neuronal depolarization‐induced rebalance of mitochondrial motility between anterograde versus retrograde transport underlies the proper formation of axonal branches. We found that the depolarization‐induced branch formation was blocked by the uncoupler p‐trifluoromethoxyphenylhydrazone, which suggests that mitochondria‐derived ATP mediates the observed phenomena. Real‐time analysis of mitochondrial movement defined the molecular mechanisms by showing that the pharmacological activation of AMP‐activated protein kinase (AMPK) after depolarization increased anterograde transport of mitochondria into axons. Simultaneous imaging of axonal morphology and mitochondrial distribution revealed that mitochondrial localization preceded the emergence of axonal branches. Moreover, the higher probability of mitochondrial localization was correlated with the longer lifetime of axon branches. We qualitatively confirmed that neuronal ATP levels decreased immediately after depolarization and found that the phosphorylated form of AMPK was increased. Thus, this study identifies a novel role for AMPK in the transport of axonal mitochondria that underlie the neuronal activity‐dependent formation of axon branches. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 557–573, 2014     

Receptor tyrosine kinases: from biology to pathology

Receptor tyrosine kinases (RTKs) are transmembrane proteins involved in the control of fundamental cellular processes in metazoans. RTKs possess a general structure that includes an extracellular domain, a transmembrane domain and a highly conserved tyrosine kinase domain. RTKs are classified according to their variable extracellular ligand-binding domain. Studies of human RTK members have yielded a wealth of information elucidating their importance. Improper functioning of these enzymes due to mutations, mainly in the kinase domain, is often manifested in various human diseases and is known to be involved in several types of cancer. Here we summarize most of human RTKs, their cognate ligands, as well as related diseases and discuss the eventual use of certain RTKs as new therapeutic targets.