Showing papers in "Biochemical Journal in 2013"

Crystal structures of human cholinesterases in complex with huprine W and tacrine: elements of specificity for anti-Alzheimer's drugs targeting acetyl- and butyryl-cholinesterase.

Abstract: The multifunctional nature of Alzheimer's disease calls for MTDLs (multitarget-directed ligands) to act on different components of the pathology, like the cholinergic dysfunction and amyloid aggregation. Such MTDLs are usually on the basis of cholinesterase inhibitors (e.g. tacrine or huprine) coupled with another active molecule aimed at a different target. To aid in the design of these MTDLs, we report the crystal structures of hAChE (human acetylcholinesterase) in complex with FAS-2 (fasciculin 2) and a hydroxylated derivative of huprine (huprine W), and of hBChE (human butyrylcholinesterase) in complex with tacrine. Huprine W in hAChE and tacrine in hBChE reside in strikingly similar positions highlighting the conservation of key interactions, namely, π-π/cation-π interactions with Trp86 (Trp82), and hydrogen bonding with the main chain carbonyl of the catalytic histidine residue. Huprine W forms additional interactions with hAChE, which explains its superior affinity: the isoquinoline moiety is associated with a group of aromatic residues (Tyr337, Phe338 and Phe295 not present in hBChE) in addition to Trp86; the hydroxyl group is hydrogen bonded to both the catalytic serine residue and residues in the oxyanion hole; and the chlorine substituent is nested in a hydrophobic pocket interacting strongly with Trp439. There is no pocket in hBChE that is able to accommodate the chlorine substituent.

Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis

Abstract: Both Met (methionine) and SAM ( S -adenosylmethionine), the activated form of Met, participate in a number of essential metabolic pathways in plants. The subcellular compartmentalization of Met fluxes will be discussed in the present review with respect to regulation and communication with the sulfur assimilation pathway, the network of the aspartate-derived amino acids and the demand for production of SAM. SAM enters the ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for the majority of methylation reactions required for plant growth and development. The multiple essential roles of SAM require regulation of its synthesis, recycling and distribution to sustain these different pathways. A particular focus of the present review will be on the function of recently identified genes of the Met salvage cycle or Yang cycle and the importance of the Met salvage cycle in the metabolism of MTA (5′-methylthioadenosine). MTA has the potential for product inhibition of ethylene, nicotianamine and polyamine biosynthesis which provides an additional link between these pathways. Interestingly, regulation of Met cycle genes was found to differ between plant species as shown for Arabidopsis thaliana and Oryza sativa .

Wnt/β-catenin signalling: from plasma membrane to nucleus

Abstract: Wnt/β-catenin signalling plays essential roles in embryonic development as well as tissue homoeostasis in adults. Thus abnormal regulation of Wnt/β-catenin signalling is linked to a variety of human diseases, including cancer, osteoporosis and Alzheimer9s disease. Owing to the importance of Wnt signalling in a wide range of biological fields, a better understanding of its precise mechanisms could provide fundamental insights for therapeutic applications. Although many studies have investigated the regulation of Wnt/β-catenin signalling, our knowledge remains insufficient due to the complexity and diversity of Wnt signalling. It is generally accepted that the identification of novel regulators and their functions is a prerequisite to fully elucidating the regulation of Wnt/β-catenin signalling. Recently, several novel modulators of Wnt signalling have been determined through multiple genetic and proteomic approaches. In the present review, we discuss the mechanistic regulation of Wnt/β-catenin signalling by focusing on the roles of these novel regulators.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

Abstract: NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

Inositol pyrophosphates: between signalling and metabolism

Abstract: The present review will explore the insights gained into inositol pyrophosphates in the 20 years since their discovery in 1993. These molecules are defined by the presence of the characteristic ‘high energy’ pyrophosphate moiety and can be found ubiquitously in eukaryotic cells. The enzymes that synthesize them are similarly well distributed and can be found encoded in any eukaryote genome. Rapid progress has been made in characterizing inositol pyrophosphate metabolism and they have been linked to a surprisingly diverse range of cellular functions. Two decades of work is now beginning to present a view of inositol pyrophosphates as fundamental, conserved and highly important agents in the regulation of cellular homoeostasis. In particular it is emerging that energy metabolism, and thus ATP production, is closely regulated by these molecules. Much of the early work on these molecules was performed in the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum , but the development of mouse knockouts for IP6K1 and IP6K2 [IP6K is IP 6 (inositol hexakisphosphate) kinase] in the last 5 years has provided very welcome tools to better understand the physiological roles of inositol pyrophosphates. Another recent innovation has been the use of gel electrophoresis to detect and purify inositol pyrophosphates. Despite the advances that have been made, many aspects of inositol pyrophosphate biology remain far from clear. By evaluating the literature, the present review hopes to promote further research in this absorbing area of biology.

The glyoxalase pathway: the first hundred years... and beyond.

Abstract: The discovery of the enzymatic formation of lactic acid from methylglyoxal dates back to 1913 and was believed to be associated with one enzyme termed ketonaldehydemutase or glyoxalase, the latter designation prevailed. However, in 1951 it was shown that two enzymes were needed and that glutathione was the required catalytic co-factor. The concept of a metabolic pathway defined by two enzymes emerged at this time. Its association to detoxification and anti-glycation defence are its presently accepted roles, since methylglyoxal exerts irreversible effects on protein structure and function, associated with misfolding. This functional defence role has been the rationale behind the possible use of the glyoxalase pathway as a therapeutic target, since its inhibition might lead to an increased methylglyoxal concentration and cellular damage. However, metabolic pathway analysis showed that glyoxalase effects on methylglyoxal concentration are likely to be negligible and several organisms, from mammals to yeast and protozoan parasites, show no phenotype in the absence of one or both glyoxalase enzymes. The aim of the present review is to show the evolution of thought regarding the glyoxalase pathway since its discovery 100 years ago, the current knowledge on the glyoxalase enzymes and their recognized role in the control of glycation processes.

Amino acid sensing in dietary-restriction-mediated longevity: roles of signal-transducing kinases GCN2 and TOR

Abstract: DR (dietary restriction), or reduced food intake without malnutrition, is associated with extended longevity, improved metabolic fitness and increased stress resistance in a wide range of organisms. DR is often referred to as calorie restriction, implying that reduced energy intake is responsible for its widespread and evolutionarily conserved benefits. However, recent data indicate dietary amino acid restriction as a key mediator of DR benefits. In fruitflies, an imbalance in essential amino acid intake is thought to underlie longevity benefits of DR. In mammals, reduced dietary protein or essential amino acid intake can extend longevity, improve metabolic fitness and increase stress resistance. In the present paper we review two evolutionarily conserved signal transduction pathways responsible for sensing amino acid levels. The eIF2α (eukaryotic initiation factor 2α) kinase GCN2 (general amino acid control non-derepressible 2) senses the absence of one or more amino acids by virtue of direct binding to uncharged cognate tRNAs. The presence of certain amino acids, such as leucine, permits activation of the master growth regulating kinase TOR (target of rapamycin). These two signal transduction pathways react to amino acid deprivation by inhibiting general protein translation while at the same time increasing translation of specific mRNAs involved in restoring homoeostasis. Together, these pathways may contribute to the regulation of longevity, metabolic fitness and stress resistance.

Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes

Abstract: The present review focuses on the evolution of proteins and the impact of amino acid mutations on function from a structural perspective. Proteins evolve under the law of natural selection and undergo alternating periods of conservative evolution and of relatively rapid change. The likelihood of mutations being fixed in the genome depends on various factors, such as the fitness of the phenotype or the position of the residues in the three-dimensional structure. For example, co-evolution of residues located close together in three-dimensional space can occur to preserve global stability. Whereas point mutations can fine-tune the protein function, residue insertions and deletions (‘decorations’ at the structural level) can sometimes modify functional sites and protein interactions more dramatically. We discuss recent developments and tools to identify such episodic mutations, and examine their applications in medical research. Such tools have been tested on simulated data and applied to real data such as viruses or animal sequences. Traditionally, there has been little if any cross-talk between the fields of protein biophysics, protein structure–function and molecular evolution. However, the last several years have seen some exciting developments in combining these approaches to obtain an in-depth understanding of how proteins evolve. For example, a better understanding of how structural constraints affect protein evolution will greatly help us to optimize our models of sequence evolution. The present review explores this new synthesis of perspectives.

The CUL3–KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction

Abstract: The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.

YB-1: oncoprotein, prognostic marker and therapeutic target?

Abstract: Hanahan and Weinberg have proposed the 'hallmarks of cancer' to cover the biological changes required for the development and persistence of tumours [Hanahan and Weinberg (2011) Cell 144, 646-674]. We have noted that many of these cancer hallmarks are facilitated by the multifunctional protein YB-1 (Y-box-binding protein 1). In the present review we evaluate the literature and show how YB-1 modulates/regulates cellular signalling pathways within each of these hallmarks. For example, we describe how YB-1 regulates multiple proliferation pathways, overrides cell-cycle check points, promotes replicative immortality and genomic instability, may regulate angiogenesis, has a role in invasion and metastasis, and promotes inflammation. We also argue that there is strong and sufficient evidence to suggest that YB-1 is an excellent molecular marker of cancer progression that could be used in the clinic, and that YB-1 could be a useful target for cancer therapy.

Structures and target recognition modes of PDZ domains: recurring themes and emerging pictures.

Abstract: PDZ domains are highly abundant protein-protein interaction modules and are often found in multidomain scaffold proteins. PDZ-domain-containing scaffold proteins regulate multiple biological processes, including trafficking and clustering receptors and ion channels at defined membrane regions, organizing and targeting signalling complexes at specific cellular compartments, interfacing cytoskeletal structures with membranes, and maintaining various cellular structures. PDZ domains, each with ~90-amino-acid residues folding into a highly similar structure, are best known to bind to short C-terminal tail peptides of their target proteins. A series of recent studies have revealed that, in addition to the canonical target-binding mode, many PDZ-target interactions involve amino acid residues beyond the regular PDZ domain fold, which we refer to as extensions. Such extension sequences often form an integral structural and functional unit with the attached PDZ domain, which is defined as a PDZ supramodule. Correspondingly, PDZ-domain-binding sequences from target proteins are frequently found to require extension sequences beyond canonical short C-terminal tail peptides. Formation of PDZ supramodules not only affords necessary binding specificities and affinities demanded by physiological functions of PDZ domain targets, but also provides regulatory switches to be built in the PDZ-target interactions. At the 20th anniversary of the discovery of PDZ domain proteins, we try to summarize structural features and target-binding properties of such PDZ supramodules emerging from studies in recent years.

Protein kinase C pharmacology: refining the toolbox.

Abstract: PKC (protein kinase C) has been in the limelight since the discovery three decades ago that it acts as a major receptor for the tumour-promoting phorbol esters. Phorbol esters, with their potent ability to activate two of the three classes of PKC isoenzymes, have remained the best pharmacological tool for directly modulating PKC activity. However, with the discovery of other phorbol ester-responsive proteins, the advent of various small-molecule and peptide modulators, and the need to distinguish isoenzyme-specific activity, the pharmacology of PKC has become increasingly complex. Not surprisingly, many of the compounds originally touted as direct modulators of PKC have subsequently been shown to hit many other cellular targets and, in some cases, not even directly modulate PKC. The complexities and reversals in PKC pharmacology have led to widespread confusion about the current status of the pharmacological tools available to control PKC activity. In the present review, we aim to clarify the cacophony in the literature regarding the current state of bona fide and discredited cellular PKC modulators, including activators, small-molecule inhibitors and peptides, and also address the use of genetically encoded reporters and of PKC mutants to measure the effects of these drugs on the spatiotemporal dynamics of signalling by specific isoenzymes.

PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress

Abstract: Vascular injury and chronic arterial diseases result in exposure of VSMCs (vascular smooth muscle cells) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation, but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with PDGF (platelet-derived growth factor)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and up-regulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3 (microtubule-associated protein light chain 3 α; also known as MAP1LC3A)-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1 or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 stabilized α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix, and abrogated VSMC hyperproliferation and migration. Treatment of cells with PDGF prevented protein damage and cell death caused by exposure to the lipid peroxidation product 4-hydroxynonenal. The results of the present study demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under the conditions of high oxidative stress found to occur in vascular lesions.

Promiscuity as a functional trait: intrinsically disordered regions as central players of interactomes

Abstract: Because of their pervasiveness in eukaryotic genomes and their unique properties, understanding the role that ID (intrinsically disordered) regions in proteins play in the interactome is essential for gaining a better understanding of the network. Especially critical in determining this role is their ability to bind more than one partner using the same region. Studies have revealed that proteins containing ID regions tend to take a central role in protein interaction networks; specifically, they act as hubs, interacting with multiple different partners across time and space, allowing for the co-ordination of many cellular activities. There appear to be three different modules within ID regions responsible for their functionally promiscuous behaviour: MoRFs (molecular recognition features), SLiMs (small linear motifs) and LCRs (low complexity regions). These regions allow for functionality such as engaging in the formation of dynamic heteromeric structures which can serve to increase local activity of an enzyme or store a collection of functionally related molecules for later use. However, the use of promiscuity does not come without a cost: a number of diseases that have been associated with ID-containing proteins seem to be caused by undesirable interactions occurring upon altered expression of the ID-containing protein.

The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system.

Abstract: The compound BAY 11-7082 inhibits IκBα [inhibitor of NF-κB (nuclear factor κB)α] phosphorylation in cells and has been used to implicate the canonical IKKs (IκB kinases) and NF-κB in >350 publications In the present study we report that BAY 11-7082 does not inhibit the IKKs, but suppresses their activation in LPS (lipopolysaccharide)-stimulated RAW macrophages and IL (interleukin)-1-stimulated IL-1R (IL-1 receptor) HEK (human embryonic kidney)-293 cells BAY 11-7082 exerts these effects by inactivating the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys63-linked and linear polyubiquitin chains BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7 by forming a covalent adduct with their reactive cysteine residues via Michael addition at the C3 atom of BAY 11-7082, followed by the release of 4-methylbenzene-sulfinic acid BAY 11-7082 stimulated Lys48-linked polyubiquitin chain formation in cells and protected HIF1α (hypoxia-inducible factor 1α) from proteasomal degradation, suggesting that it inhibits the proteasome The results of the present study indicate that the anti-inflammatory effects of BAY 11-7082, its ability to induce B-cell lymphoma and leukaemic T-cell death and to prevent the recruitment of proteins to sites of DNA damage are exerted via inhibition of components of the ubiquitin system and not by inhibiting NF-κB

Elevated SGK1 predicts resistance of breast cancer cells to Akt inhibitors

Abstract: The majority of human cancers harbour mutations promoting activation of the Akt protein kinase, and Akt inhibitors are being evaluated in clinical trials. An important question concerns the understanding of the innate mechanisms that confer resistance of tumour cells to Akt inhibitors. SGK (serum- and glucocorticoid-regulated kinase) is closely related to Akt and controlled by identical upstream regulators {PI3K (phosphoinositide 3-kinase), PDK1 (phosphoinositide-dependent kinase 1) and mTORC2 [mTOR (mammalian target of rapamycin) complex 2]}. Mutations that trigger activation of Akt would also stimulate SGK. Moreover, Akt and SGK possess analogous substrate specificities and are likely to phosphorylate overlapping substrates to promote proliferation. To investigate whether cancers possessing high SGK activity could possess innate resistance to Akt-specific inhibitors (that do not target SGK), we analysed SGK levels and sensitivity of a panel of breast cancer cells towards two distinct Akt inhibitors currently in clinical trials (AZD5363 and MK-2206). This revealed a number of Akt-inhibitor-resistant lines displaying markedly elevated SGK1 that also exhibited significant phosphorylation of the SGK1 substrate NDRG1 [N-Myc (neuroblastoma-derived Myc) downstream-regulated gene 1]. In contrast, most Akt-inhibitor-sensitive cell lines displayed low/undetectable levels of SGK1. Intriguingly, despite low SGK1 levels, several Akt-inhibitor-sensitive cells showed marked NDRG1 phosphorylation that was, unlike in the resistant cells, suppressed by Akt inhibitors. SGK1 knockdown markedly reduced proliferation of Akt-inhibitor-resistant, but not -sensitive, cells. Furthermore, treatment of Akt-inhibitor-resistant cells with an mTOR inhibitor suppressed proliferation and led to inhibition of SGK1. The results of the present study suggest that monitoring SGK1 levels as well as responses of NDRG1 phosphorylation to Akt inhibitor administration could have a use in predicting the sensitivity of tumours to compounds that target Akt. Our findings highlight the therapeutic potential that SGK inhibitors or dual Akt/SGK inhibitors might have for treatment of cancers displaying elevated SGK activity.

Evolution of intracellular compartmentalization.

Abstract: Cells compartmentalize their biochemical functions in a variety of ways, notably by creating physical barriers that separate a compartment via membranes or proteins. Eukaryotes have a wide diversity of membrane-based compartments, many that are lineage- or tissue-specific. In recent years, it has become increasingly evident that membrane-based compartmentalization of the cytosolic space is observed in multiple prokaryotic lineages, giving rise to several types of distinct prokaryotic organelles. Endosymbionts, previously believed to be a hallmark of eukaryotes, have been described in several bacteria. Protein-based compartments, frequent in bacteria, are also found in eukaryotes. In the present review, we focus on selected intracellular compartments from each of these three categories, membrane-based, endosymbiotic and protein-based, in both prokaryotes and eukaryotes. We review their diversity and the current theories and controversies regarding the evolutionary origins. Furthermore, we discuss the evolutionary processes acting on the genetic basis of intracellular compartments and how those differ across the domains of life. We conclude that the distinction between eukaryotes and prokaryotes no longer lies in the existence of a compartmentalized cell plan, but rather in its complexity.

The CARD plays a critical role in ASC foci formation and inflammasome signalling.

Abstract: The ASC (apoptosis speck-like protein) is a key component of multimeric protein complexes that mediate inflammation and host defence. Comprising a PYD (Pyrin) domain and a CARD (caspase activation and recruitment domain), ASC functions downstream of NLRs (nucleotide-binding domain, leucine-rich repeat-containing receptors) and AIM2 (absent in melanoma 2) through the formation of supramolecular structures termed inflammasomes. However, the mechanism underlying ASC signalling and its dependency on oligomeric arrangements in inflammasome formation remain poorly understood. When expressed in cells, ASC forms discrete foci (called 'specks') typically with one speck per cell. We employed a BiFC (bimolecular fluorescence complementation) system to investigate and visualize ASC foci formation in living cells. We demonstrated that the CARD of ASC plays a central role in ASC inflammasome assembly, representing the minimal unit capable of forming foci in conjunction with the caspase 1 CARD. Mutational studies point to multiple surfaces on the ASC CARD and two predominant areas on the caspase 1 CARD mediating the formation of ASC/caspase 1 foci. The lack of foci formation for ASC CARD mutants correlates with a loss of IL-1β (interleukin 1β) processing in response to NLRP (NLR family, PYD domain-containing) 3 or AIM2 agonists in RAW264.7 cell reconstitution assays. Analogously, we show that productive formation of the Salmonella typhimurium-induced NLRC4 (NLR family CARD domain-containing protein 4) inflammasome is dependent on ASC-CARD-mediated platform formation. Thus the results of the present study depict a central role of CARDs in the formation of ASC signalling platforms and provide an important tool for investigation of CARD-dependent networks.

Identification of novel candidate phosphatidic acid-binding proteins involved in the salt-stress response of Arabidopsis thaliana roots.

Abstract: PA (phosphatidic acid) is a lipid second messenger involved in an array of processes occurring during a plant's life cycle. These include development, metabolism, and both biotic and abiotic stress responses. PA levels increase in response to salt, but little is known about its function in the earliest responses to salt stress. In the present study we have combined an approach to isolate peripheral membrane proteins of Arabidopsis thaliana roots with lipid-affinity purification, to identify putative proteins that interact with PA and are recruited to the membrane in response to salt stress. Of the 42 putative PA-binding proteins identified by MS, a set of eight new candidate PA-binding proteins accumulated at the membrane fraction after 7 min of salt stress. Among these were CHC (clathrin heavy chain) isoforms, ANTH (AP180 N-terminal homology) domain clathrin-assembly proteins, a putative regulator of potassium transport, two ribosomal proteins, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and a PI (phosphatidylinositol) 4-kinase. PA binding and salt-induced membrane recruitment of GAPDH and CHC were confirmed by Western blot analysis of the cellular fractions. In conclusion, the approach of the present study is an effective way to isolate biologically relevant lipid-binding proteins and provides new leads in the study of PA-mediated salt-stress responses in roots.

Brown adipose tissue: development, metabolism and beyond

Abstract: Obesity represents a major risk factor for the development of several of our most common medical conditions, including Type 2 diabetes, dyslipidaemia, non-alcoholic fatty liver, cardiovascular disease and even some cancers. Although increased fat mass is the main feature of obesity, not all fat depots are created equal. Adipocytes found in white adipose tissue contain a single large lipid droplet and play well-known roles in energy storage. By contrast, brown adipose tissue is specialized for thermogenic energy expenditure. Owing to its significant capacity to dissipate energy and regulate triacylglycerol (triglyceride) and glucose metabolism, and its demonstrated presence in adult humans, brown fat could be a potential target for the treatment of obesity and metabolic syndrome. Undoubtedly, fundamental knowledge about the formation of brown fat and regulation of its activity is imperatively needed to make such therapeutics possible. In the present review, we integrate the recent advancements on the regulation of brown fat formation and activity by developmental and hormonal signals in relation to its metabolic function.

The cell biology of prion-like spread of protein aggregates: mechanisms and implication in neurodegeneration.

Abstract: The misfolding and aggregation of specific proteins is a common hallmark of many neurodegenerative disorders, including highly prevalent illnesses such as Alzheimer's and Parkinson's diseases, as well as rarer disorders such as Huntington's and prion diseases. Among these, only prion diseases are 'infectious'. By seeding misfolding of the PrP(C) (normal conformer prion protein) into PrP(Sc) (abnormal disease-specific conformation of prion protein), prions spread from the periphery of the body to the central nervous system and can also be transmitted between individuals of the same or different species. However, recent exciting data suggest that the transmissibility of misfolded proteins within the brain is a property that goes way beyond the rare prion diseases. Evidence indicates that non-prion aggregates [tau, α-syn (α-synuclein), Aβ (amyloid-β) and Htt (huntingtin) aggregates] can also move between cells and seed the misfolding of their normal conformers. These findings have enormous implications. On the one hand they question the therapeutical use of transplants, and on the other they indicate that it may be possible to bring these diseases to an early arrest by preventing cell-to-cell transmission. To better understand the prion-like spread of these protein aggregates it is essential to identify the underlying cellular and molecular factors. In the present review we analyse and discuss the evidence supporting prion-like spreading of amyloidogenic proteins, especially focusing on the cellular and molecular mechanisms and their significance.

Transcriptional up-regulation of ULK1 by ATF4 contributes to cancer cell survival.

Abstract: Hypoxia in the microenvironment of many solid tumours is an important determinant of malignant progression. The ISR (integrated stress response) protects cells from the ER (endoplasmic reticulum) stress caused by severe hypoxia. Likewise, autophagy is a mechanism by which cancer cells can evade hypoxic cell death. In the present paper we report that the autophagy-initiating kinase ULK1 (UNC51-like kinase 1) is a direct transcriptional target of ATF4 (activating transcription factor 4), which drives the expression of ULK1 mRNA and protein in severe hypoxia and ER stress. We demonstrate that ULK1 is required for autophagy in severe hypoxia and that ablation of ULK1 causes caspase-3/7-independent cell death. Furthermore, we report that ULK1 expression is associated with a poor prognosis in breast cancer. Collectively, the findings of the present study identify transcriptional up-regulation of ULK1 as a novel arm of the ISR, and suggest ULK1 as a potentially effective target for cancer therapy.

CRISPR interference: a structural perspective

Abstract: CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as ‘interference’. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system.

Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5

Abstract: WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3–9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein–protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.

Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis

Abstract: Programmed necrosis or necroptosis is controlled by the action of two serine/threonine kinases, RIP1 (receptor-interacting serine/threonine protein kinase 1; also known as RIPK1) and RIP3. The phosphorylation of RIP1 and RIP3 is critical for assembly of the necrosome, an amyloid-like complex that initiates transmission of the pro-necrotic signal. In the present study, we used site-directed mutagenesis to systematically examine the effects of putative phosphoacceptor sites on RIP1 and RIP3 on TNF (tumour necrosis factor)-induced programmed necrosis. We found that mutation of individual serine residues in the kinase domain of RIP1 had little effect on RIP1 kinase activity and TNF-induced programmed necrosis. Surprisingly, an alanine residue substitution for Ser(89) enhanced RIP1 kinase activity and TNF-induced programmed necrosis without affecting RIP1-RIP3 necrosome formation. This indicates that Ser(89) is an inhibitory phosphoacceptor site that can dampen the pro-necrotic function of RIP1. In addition, we show that a phosphomimetic mutant of RIP3, S204D, led to programmed necrosis that was refractory to RIP1 siRNA and insensitive to necrostatin-1 inhibition. Our results show that programmed necrosis is regulated by positive and inhibitory phosphorylation events.

Mitochondrial respiratory chain complex I is inactivated by NADPH oxidase Nox4

Abstract: ROS (reactive oxygen species) generated by NADPH oxidases play an important role in cellular signal transduction regulating cell proliferation, survival and differentiation. Nox4 (NADPH oxidase 4) induces cellular senescence in human endothelial cells; however, intracellular targets for Nox4 remained elusive. In the present study, we show that Nox4 induces mitochondrial dysfunction in human endothelial cells. Nox4 depletion induced alterations in mitochondrial morphology, stabilized mitochondrial membrane potential and decreased production of H(2)O(2) in mitochondria. High-resolution respirometry in permeabilized cells combined with native PAGE demonstrated that Nox4 specifically inhibits the activity of mitochondrial electron transport chain complex I, and this was associated with a decreased concentration of complex I subunits. These data suggest a new pathway by which sustained Nox4 activity decreases mitochondrial function.

Describing sequence–ensemble relationships for intrinsically disordered proteins

Abstract: Emil Fischer proposed that enzyme specificity could be explained by shape complementarity [1, 2]. He used the metaphor of a lock and key to illustrate how the three-dimensional arrangements of atoms comprising an enzyme and its substrate could enable them to fit together and prevent non-specific catalysis. Interpreted literally, this metaphor suggests that proteins possess rigid structures that determine their functions. Fischer, however, felt that popular interpretations of his lock-and-key metaphor exceeded its scope and experimental justification [3]. Indeed, protein rigidity has proven to be unsatisfactory at explaining noncompetitive inhibition and cannot account for enzymes where binding of one reactive group increases the exposure of another [4]. The concepts of allosteric linkage [5] and induced fit require the invocation of protein conformational changes in response to the binding of an interaction partner [6]. The structure-function paradigm, nuanced to accommodate proteins that switch between discrete conformations with different shape complementarities for execution of specific functions provides visual clarity and mathematical simplicity. Going beyond structure-function relationships Advancements on scientific and technological fronts have demonstrated unequivocally that proteins can exhibit significant conformational heterogeneity. Intrinsically disordered proteins (IDPs) are at the extreme end of the heterogeneity spectrum [7-9]. They adopt ensembles of conformations in aqueous solutions for which no single structure or self-similar collection of structures provides an adequate description. By all accounts the conformational heterogeneity exhibited by IDPs is relevant for biological function [8-14]. The phrase intrinsically disordered proteins is used to imply that the amino acid sequences for this class of proteins encode a preference for heterogeneous ensembles of conformations as the thermodynamic ground state under standard physiological conditions (aqueous solutions, 150 mM monovalent salt, low concentrations of divalent ions, pH 7.0, and temperature in the 25°C – 37°C range) [9, 15]. For many IDPs, folding can be coupled to binding and they can adopt ordered structures in specific bound complexes [16-20]. The intrinsic heterogeneity in their unbound forms is reflected in their ability to adopt different folds in the context of different complexes [10]. Transcription factors represent striking examples of molecules that undergo disorder-to-order transitions in complex with their cognate DNA partners [21-24]. Highly stable complexes with DNA can make transcription factor dissociation become “unreasonably slow” when compared to the turnover time of downstream regulatory processes. Disorder in the unbound forms is proposed to be important for lowering the overall affinity, which in turn increases the off-rates of protein-DNA complexes [25]. There are a growing number of reports of “fuzzy complexes” whereby conformational heterogeneity prevails in binary and multimolecular complexes [26-28]. IDPs can also self-assemble to form ordered, supramolecular assemblies, although the degree of order within these assemblies is variable and the intermediates that seem to be obligatory for self-assembly are characterized by significant conformational heterogeneity that can be modulated to alter the mechanisms of self-assembly and the stabilities of supramolecular structures [29-38].

A broad activity screen in support of a chemogenomic map for kinase signalling research and drug discovery.

Abstract: Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of kinase inhibition across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using biochemical assays for inhibitory activity against 234 active human kinases and kinase complexes, representing all branches of the kinome tree. For our study we employed 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important as therapeutic targets and/or signal transduction regulators. Hierarchical clustering of these benchmark kinase inhibitors on the basis of their kinome activity profiles illustrates how they relate to chemical structure similarities and provides new insights into inhibitor specificity and potential applications for probing new targets. Using this broad dataset, we provide a framework for assessing polypharmacology. We not only discover likely off-target inhibitor activities and recommend specific inhibitors for existing targets, but also identify potential new uses for known small molecules.

The metalloproteases meprin α and meprin β: unique enzymes in inflammation, neurodegeneration, cancer and fibrosis

Abstract: The metalloproteases meprin α and meprin β exhibit structural and functional features that are unique among all extracellular proteases. Although meprins were discovered more than 30 years ago, their precise substrates and physiological roles have been elusive. Both enzymes were originally found to be highly expressed in kidney and intestine, which focused research on these particular tissues and associated pathologies. Only recently it has become evident that meprins exhibit a much broader expression pattern, implicating functions in angiogenesis, cancer, inflammation, fibrosis and neurodegenerative diseases. Different animal models, as well as proteomics approaches for the identification of protease substrates, have helped to reveal more precise molecular signalling events mediated by meprin activity, such as activation and release of pro-inflammatory cytokines. APP (amyloid precursor protein) is cleaved by meprin β in vivo, reminiscent of the β-secretase BACE1 (β-site APP-cleaving enzyme 1). The subsequent release of Aβ (amyloid β) peptides is thought to be the major cause of the neurodegenerative Alzheimer's disease. On the other hand, ADAM10 (a disintegrin and metalloprotease domain 10), which is the constitutive α-secretase, was shown to be activated by meprin β, which is itself shed from the cell surface by ADAM10. In skin, both meprins are overexpressed in fibrotic tumours, characterized by massive accumulation of fibrillar collagens. Indeed, procollagen III is processed to its mature form by meprin α and meprin β, an essential step in collagen fibril assembly. The recently solved crystal structure of meprin β and the unique cleavage specificity of these proteases identified by proteomics will help to generate specific inhibitors that could be used as therapeutics to target meprins under certain pathological conditions.

Mind the gap: cytochrome interactions reveal electron pathways across the periplasm of Shewanella oneidensis MR-1.

Abstract: Extracellular electron transfer is the key metabolic trait that enables some bacteria to play a significant role in the biogeochemical cycling of metals and in bioelectrochemical devices such as microbial fuel cells. In Shewanella oneidensis MR-1, electrons generated in the cytoplasm by catabolic processes must cross the periplasmic space to reach terminal oxidoreductases found at the cell surface. Lack of knowledge on how these electrons flow across the periplasmic space is one of the unresolved issues related with extracellular electron transfer. Using NMR to probe protein-protein interactions, kinetic measurements of electron transfer and electrostatic calculations, we were able to identify protein partners and their docking sites, and determine the dissociation constants. The results showed that both STC (small tetrahaem cytochrome c) and FccA (flavocytochrome c) interact with their redox partners, CymA and MtrA, through a single haem, avoiding the establishment of stable redox complexes capable of spanning the periplasmic space. Furthermore, we verified that the most abundant periplasmic cytochromes STC, FccA and ScyA (monohaem cytochrome c5) do not interact with each other and this is likely to be the consequence of negative surface charges in these proteins. This reveals the co-existence of two non-mixing redox pathways that lead to extracellular electron transfer in S. oneidensis MR-1 established through transient protein interactions.