Nat Rev Mol Cell Biol Research Articles

Healthy ageing through regulated proteostasis

During metazoan life, protein homeostasis (proteostasis) declines with age due to impaired proteasome activity. The signalling pathways activated by growth factors that regulate proteostasis and their importance to ageing are just emerging. In this issue, Liu et al report a crucial role of epidermal growth factor (EGF) in regulating Caenorhabditis elegans lifespan through the RAF/MAPK pathway. EGF signalling activates the ubiquitin–proteosome system (UPS) and represses the chaperone machinery by modulating the expression of several ageing‐related genes (gerontogenes). This strategic switch in controlling global protein turnover provides novel insights into the molecular mechanisms driving ageing in multi‐cellular organisms.

A mad partner for Shugoshin in meiosis

The centromeric cohesin protector Shugoshin (Sgo) and the spindle checkpoint protein Mad2 are known to act in distinct pathways for ensuring accurate chromosome segregation during eukaryotic cell division. In this issue of The EMBO Journal , [Orth et al (2011)][1] describe an unexpected physical interaction between vertebrate Sgo and Mad2, as well as indications of their potential mutual regulation in meiosis. [1]: #ref-7

How uniform is cAMP signaling? Focus on “Systems analysis of GLP-1 receptor signaling in pancreatic β-cells”

the goal of many cell physiologists is to ascribe cellular behavior to specific molecular signaling mechanisms. However, the reductionist experimental approach commonly used to understand molecular function often makes this task harder rather than easier to accomplish because of the complexity of

Mitochondria breathe for autophagy

Proper mitochondrial structure and function are established by continuous cycles of fusion and fission, and a unidirectional pathway of autophagy‐mediated degradation. In this issue of The EMBO Journal , Graef and Nunnari (2011) demonstrate that autophagy requires healthy mitochondria in cells under amino‐acid starvation. Defects in mitochondrial respiration cause activation of cAMP‐dependent protein kinase A (PKA), an evolutionarily conserved regulator that inhibits autophagic flux. This study reveals a crucial interplay between mitochondrial function and autophagy, and raises the possibility that deregulation in one of these processes simultaneously compromises the other to potentiate risks for various human disorders.

Arp you ready for actin in the nucleus?

Actin and actin‐related proteins (Arps) are critical components of chromatin remodelling and modifying complexes, where they are commonly found in pairs. Yet, little is known about their role in these complexes. In this issue of The EMBO Journal , Fenn et al (2011) report the first structural investigation of two nuclear Arps, the INO80 complex subunits Arp4 and Arp8. Importantly, their results also shed light on the critical role that the nucleotide state of actin may have on its interactions with the Arps.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

Abstract 4683: Inducible knockdown of DNA topoisomerase IIα affects drug sensitivity and cell growth

Abstract DNA topoisomerase IIα (Top2α) is an essential nuclear enzyme and target for important anti-cancer drugs, such as etoposide and doxorubicin (Dox). Our teniposide-resistant human leukemic lymphoblastic cells (CEM/VM-1-5) (Danks MK et al., Biochemistry 27(24):8861-8869, 1998) express reduced Top2α protein. To determine the role of Top2α in drug resistance, we knocked down Top2α in parental CEM cells by RNAi. Compared to control cells, CEMshTop2α cells were more resistant to the Top2 inhibitors, etoposide and Dox. The mechanism by which knockdown of Top2α caused resistance to Top2 inhibitors is probably caused by a reduction in Top2-DNA complexes leading to fewer broken DNA strands (Wang JC, Nat Rev Mol Cell Biol 3(6):430-440, 2002). The level of reduced Top2α expression in Top2α knockdown CEM cells was confirmed by Western blot. Importantly, we found no compensatory regulation by Top1 or Top2α overexpression under these conditions. It has been reported that knocking-out of Top2α will cause early embryonic death (Jenkins JR et al. Nucleic acids research 20(21):5587-5592, 1992), but in our cultured CEM cells, knocking Top2α down to 40% of control level had little or no impact on cell growth rate. We subsequently generated and characterized stable CEM-tTR-shTop2α cells where Top2α can be conditionally knocked down to 5% of control levels, as determined by Western blot, through a tetracycline-induced expression of short hairpin RNAs. CEM-tTR-shTop2α cells grow significantly more slowly than the control cells, which indicates that while cells can proliferate with ∼40% of Top2α expression, when Top2α is reduced by 95%, cells can apparently remain viable, but are severely impaired in their ability to grow. Present studies are focused on identifying genes that are targets of Top2α knockdown to provide insights into those genes involved in drug resistance. (Supported in part by CA40570 from NCI and in part by UIC) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4683. doi:10.1158/1538-7445.AM2011-4683

Chaperone receptors: guiding proteins to intracellular compartments

Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519-530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529-535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41-50, 2003; Qbadou et al., EMBO J 25:1836-1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.

Structure and function of a HECT domain ubiquitin‐binding site

The Rsp5 ubiquitin ligase contains a non-covalent binding site for ubiquitin within the amino-terminal lobe (N-lobe) of the HECT domain, and the X-ray crystal structure of the HECT-ubiquitin complex has been determined. Hydrophobic patch residues of ubiquitin (L8, I44, V70) were crucial for interaction with Rsp5, and amino-acid alterations at the Rsp5-binding interface resulted in defects in polyubiquitination. Our results support a model in which the N-lobe-binding site acts to localize and orient the distal end of the ubiquitin chain to promote conjugation of the next ubiquitin molecule.

AMPK and autophagy get connected

Niida et al provide the first conclusive evidence that the DNA damage checkpoint kinases Chk1 and Chk2 are bona fide tumour suppressors, also giving insight into the unique and redundant functions of these two factors in vivo.

Inducible SUMO modification of TANK alleviates its repression of TLR7 signalling

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.

Hippo Tumor Supressor Pathway: Novel Implications for the Treatment of Hepatocellular Carcinoma

Zhou D, Conrad C, Xia F, et al. (Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts). Mst1 and Mst2 maintain quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 2009;16:425–438.

A novel role for sphingolipid metabolism in oxidant-mediated skeletal muscle fatigue. Focus on “Sphingomyelinase stimulates oxidant signaling to weaken skeletal muscle and promote fatigue”

sphingolipids are a chemically diverse group of biological lipids typified by a sphingoid base backbone derived from condensation of a fatty acyl-CoA to an amino acid (typically, but not always, serine) ([24][1], [27][2]). These lipids occur throughout eukaryotic species, thus reflecting their

An Energy-Sensor Network Takes Center Stage During Endothelial Aging

See related article, pages 1384–1393 Blood vessels form a critical interface between the environment and the organism by supplying nutrients, oxygen and macromolecules to all tissues and cells in the body. Endothelial cells (ECs) line the inner surface of the hierarchically branched blood vessel network and integrate functionally into different organs to support tissue growth and function in development, physiology, and disease. In healthy postnatal tissues, ECs are mostly quiescent and rarely migrate or proliferate. However, ECs possess a remarkable phenotypic plasticity in terms of being able to proliferate rapidly in response to vascular injury, tissue ischemia, or other stress conditions. This phenotypic plasticity progressively declines when ECs age and/or are permanently exposed to metabolic (eg, hyperglycemia) and environmental stressors (eg, oxidative stress) and coincides with the development of endothelial senescence.1 Cellular senescence is a stress response that is characterized by a robust inhibition of cell proliferation, which often becomes irreversible and independent of the initial stress signal.2 Senescence can be observed in most mammalian cells after extended propagation in culture and is related to the attrition of telomeres after repetitive cycles of cell divisions.2 In addition to telomere shortening, other stressors such as DNA damage, genomic instability, and oxidative stress can induce a similar growth arrest and trigger senescence.2 Senescent cells display characteristic alterations in cell morphology and gene expression that may weaken cellular functions. In ECs, these changes cause a phenotype that is proinflammatory, prothrombotic, and proatherosclerotic, which also negatively affects the vasodilatory, angiogenic, and regenerative properties of ECs and, thus, accelerates the development of several cardiovascular diseases.1 Despite advances in understanding the molecular mechanisms of endothelial senescence and vascular aging, the signaling networks governing the development and progression of EC senescence remain less well defined. In this issue of Circulation Research …

Wnt Signaling Molecules in Left Ventricular Remodeling

Under acute or chronic stresses, the adult heart undergoes a remodeling process that involves cardiomyocyte hypertrophy accompanied by apoptosis, necrosis, and fibrosis that lead to impaired cardiac contractility. The role of endogenous regeneration in this process is currently under investigation. Sustained deleterious stimuli will lead to a decompensated form of hypertrophy often culminating in heart failure.1 This form of hypertrophy is often referred to as “maladaptive.” When dealing with hypertrophy, it appears important to distinguish between the term being used on the cellular and molecular level (enlargement of individual cardiomyocytes and re-expression of fetal/embryonic genes) and the organ level (increased heart weight, left ventricular wall thickness, and functional diastolic and systolic impairment). In our view, these processes are certainly linked but not identical. Hypertrophy on the organ levels summarizes several independent cellular and molecular processes (see below), where cardiomyocyte growth is not necessarily the most important. Independent of its origin, cardiac hypertrophy is associated with alterations in cardiac geometry, mass, architecture, and function controlled by a complex network of interconnected and abundant signal-transduction pathways.2 New signaling molecules are emerging as possible targets to specifically attenuate maladaptive hypertrophy. Pathological, stress-induced growth of cardiomyocytes was shown to depend on Wnt/β-catenin nuclear signaling rather than its adhesive function in cell adhesion. However, the specificity of the cell type and the molecular mechanisms governing the Wnt signaling–dependent changes are currently unknown.3 In this issue of the Hypertension , the study by Malekar et al4 provides new evidences concerning the …

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca2+ going local?

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca²⁺ going local?

Apoptosis: it's BAK to VDAC

VDAC's role in Bak‐dependent mitochondrial outer membrane permeabilization (MOMP) is controversial. Gordon Shore makes sense of it all in light of an EMBO reports paper that shows that VDAC mediates mitochondrial recruitment of Bak, thereby controlling tBid‐induced MOMP.

Mitogen-Activated Protein Kinases as Biomarkers of Hypertension or Cardiac Pressure Overload

In hypertension, left ventricular hypertrophy is initially a useful compensatory process that represents an adaptation to increased ventricular wall stress. It is also one of the first steps toward overt clinical disease. Several changes in myocardial structure characterize hypertensive or pressure-overload heart disease that induces myocardial remodeling (eg, enhanced cardiomyocyte growth, excessive cardiomyocyte necrosis/apoptosis, accumulation of interstitial and perivascular collagen fibers, or disruption of the endomysial and perimysial collagen network).1 In the long term, these cellular changes, if untreated, will deteriorate left ventricular function and facilitate the development of heart failure. The growth and survival of adult cardiomyocytes, smooth muscle cells, and macrophages are regulated by extracellular ligands, growth factors, and cytokines that bind to cell-surface receptors and activate intracellular signal transduction cascades. These signaling pathways control essential processes in all eukaryotic cells, including gene transcription, protein translation, cytoskeletal remodeling, endocytosis, cell metabolism, cell proliferation, and survival.2 One of the best-described signal transmission systems activated by pressure overload involves multiple cascades of protein phosphorylation by the mitogen activated protein kinase (MAPK) family, with diverse roles in a broad range of physiological functions.3 The MAPK signaling pathway sequences ultimately result in the dual phosphorylation and activation of terminal kinases, such as p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERK1/2 and ERK5), which are involved in different cellular responses, including pressure-overload–induced cardiac hypertrophy.4 MAPK cascades are triple-kinase pathways that include an MAPK kinase kinase, an MAPK kinase, and a terminal MAPK. MAPK cascades may be …

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-kappaB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-kappaB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65(-/-) murine fibroblasts immortalize at considerably faster rates than RelA/p65(+/+) cells. The ability of RelA/p65(-/-) fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65(-/-) fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-kappaB. Altogether, our findings present a fresh perspective on the role of NF-kappaB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.