Protein SUMOylation and phase separation: partners in stress?

Protein SUMOylation is one of the most prevalent post-translational modifications (PTMs) and important for maintaining cellular homeostasis in response to various cellular stresses. Emerging evidence reveals the role of liquid–liquid phase separation (LLPS)/biomolecular condensates in cellular SUMOylation, potentially solving a puzzle regarding the cellular mechanism of SUMOylation regulation.     

Diacylglycerol kinases: why so many of them?

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. In addition to the C1 domains (protein kinase C-like zinc finger structures) conserved commonly in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain and ankyrin repeats. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways conducting development, neural and immune responses, cytoskeleton reorganization and carcinogenesis. Moreover, there has been rapidly growing evidence indicating that individual DGK isoforms exert their specific roles through interactions with unique partner proteins such as protein kinase Cs, Ras guanyl nucleotide-releasing protein, chimaerins and phosphatidylinositol-4-phosphate 5-kinase. Therefore, an emerging paradigm for DGK is that the individual DGK isoforms assembled in their own signaling complexes should carry out spatio-temporally segregated tasks for a wide range of biological processes via regulating local, but not global, concentrations of DAG and/or PA.     

Inhibitors of c-Jun N-terminal kinases: JuNK no more?

The c-Jun N-terminal kinases (JNKs) have been the subject of intense interest since their discovery in the early 1990s. Major research programs have been directed to the screening and/or design of JNK-selective inhibitors and testing their potential as drugs. We begin this review by considering the first commercially-available JNK ATP-competitive inhibitor, SP600125. We focus on recent studies that have evaluated the actions of SP600125 in lung, brain, kidney and liver following exposure to a range of stress insults including ischemia/reperfusion. In many but not all cases, SP600125 administration has proved beneficial. JNK activation can also follow infection, and we next consider recent examples that demonstrate the benefits of SP600125 administration in viral infection. Additional ATP-competitive JNK inhibitors have now been described following high throughput screening of small molecule libraries, but information on their use in biological systems remains limited and thus these inhibitors will require further evaluation. Peptide substrate-competitive ATP-non-competitive inhibitors of JNK have also now been described, and we discuss the recent advances in the use of JNK inhibitory peptides in the treatment of neuronal death, diabetes and viral infection. We conclude by raising a number of questions that should be considered in the quest for JNK-specific inhibitors.     

Ant attendance in aphids: why different degrees of myrmecophily?

1. Aphids show a range of associations with ants from nonattendance to obligate myrmecophily. Aphis fabae cirsiiacanthoides is facultatively associated with ants, while Symydobius oblongus is an obligate myrmecophile. The selection pressures that have shaped these associations are unknown. 2. The consequences for these aphids of their different degrees of associations with ants were determined, in terms of costs and benefits to individuals and colonies in laboratory and field experiments. In the laboratory, individuals of A. f. cirsiiacanthoides performed worse and those of Symydobius oblongus performed better when attended by the ant Lasius niger than when unattended. For example, when ant-attended, A. f. cirsiiacanthoides developed more slowly, were smaller, and invested less in gonads, whereas S. oblongus developed more quickly, were larger, and invested more in gonads. In addition, the ant regulated the population size of S. oblongus to an average of 50–70 individuals per birch sapling by removing aphids, but did not regulate the population size of A. f. cirsiiacanthoides. 3. Under field conditions, ant-attended colonies of both A. f. cirsiiacanthoides and S. oblongus achieved higher peak numbers and lasted longer, and ant-attended colonies of A. f. cirsiiacanthoides produced more alate dispersers than unattended colonies. 4. The implications of divergent selection pressures for the development of myrmecophily in aphids are discussed.     

Protein kinase C – A family of protein kinases, allosteric effectors or both?

The nucleotide dependence of PKC priming phosphorylations has provided compelling evidence for an ATP-induced conformational change to the kinase domain. This has various implications for the manner in which kinase inactive dominant negative PKC mutants work and also predicts that those inactivating mutations that do not lead to loss of priming may provide a more selective route to dominant negative mutants. It might be argued that this is also the case for other kinases and that caution should be employed in considering the dominant properties of the lysine mutant forms. In fact this pattern of behaviour is shared to some extent, with very clear examples of nucleotide pocket directed inhibitors triggering related phosphorylations and responses in other kinases. Alongside these cautionary tales, the functional implications for pseudokinases are clear, so that ATP binding within a suitably conserved nucleotide pocket can trigger conformational change and impact on the scaffolding function of that pseudokinase – here the kinase itself is simply an “ATP effector”.     

Mitochondrial Protein Import: A Matter of Death?

Mitochondria play a central role not only in energy generation but also for apoptosis. A key step in mitochondrial apoptosis is the release of mitochondrial proteins, most importantly cytochrome c. This release is orchestrated by the pro- and anti-apoptotic members of the Bcl-2 protein family. The functions of these Bcl-2 family members are clear in terms of order and of principle: the pro-apoptotic BH3-only protein group contains the triggers, which cause the activation of the effectors Bax and Bak, while the anti-apoptotic Bcl-2-like proteins prevent this activation. However, the molecular details are still insufficiently clear and the proposed models have certain gaps and are partly contradictory. We have recently presented evidence that targeting to mitochondria of at least one BH3-only protein is essential for its pro-apoptotic functions. Here we discuss how this mechanism might fit into and expand existing models and speculate about the potential implications of this finding.     

Protein arginine methyltransferases: guardians of the Arg?

The recent discovery of enzymes that convert methylated arginine residues in proteins to citrulline has catapulted arginine methylation into the attention of cell-signaling researchers. Long considered a rather static post-translational modification of marginal interest, it seems that arginine methylation has now joined the group of signaling pathways that operate via pairs of antagonistic enzymes. However, many questions remain unanswered, especially concerning the removal mechanism and its implication for the physiological role of arginine methylation. I propose that, in addition to the broadly discussed function as regulator of protein activity, arginine methylation might serve a second purpose: protection of arginine residues against attack by endogenous reactive dicarbonyl agents, such as methylglyoxal, which are natural by-products of normal metabolic pathways. Inefficient detoxification of these highly cytotoxic compounds results in inactivation of proteins that is causally linked to diabetes, cancer, neurodegenerative diseases and pathophysiologies of aging. This new concept of 'arginine protection' might have far-reaching implications for the development of drugs that exploit a natural protection mechanism for medical purposes.     

The taxonomy of multivalent orientation: six modes of alternate or one?

Unlike adjacent I and II, alternate I and II orientations of interchange and tetrasome quadrivalents can be considered extremes within one particular population of orientations (alternate) and thus lack sufficient distinction to justify a separate taxonomic status. Within the population up to six types may be distinguished, but the biological significance of this distinction is small.     

Measurement of six degrees of freedom head kinematics in impact conditions employing six accelerometers and three angular rate sensors (6aω configuration)

The ability to measure six degrees of freedom (6 DOF) head kinematics in motor vehicle crash conditions is important for assessing head-neck loads as well as brain injuries. A method for obtaining accurate 6 DOF head kinematics in short duration impact conditions is proposed and validated in this study. The proposed methodology utilizes six accelerometers and three angular rate sensors (6aω configuration) such that an algebraic equation is used to determine angular acceleration with respect to the body-fixed coordinate system, and angular velocity is measured directly rather than numerically integrating the angular acceleration. Head impact tests to validate the method were conducted using the internal nine accelerometer head of the Hybrid III dummy and the proposed 6aω scheme in both low (2.3?m/s) and high (4.0?m/s) speed impact conditions. The 6aω method was compared with a nine accelerometer array sensor package (NAP) as well as a configuration of three accelerometers and three angular rate sensors (3aω), both of which have been commonly used to measure 6 DOF kinematics of the head for assessment of brain and neck injuries. The ability of each of the three methods (6aω, 3aω, and NAP) to accurately measure 6 DOF head kinematics was quantified by calculating the normalized root mean squared deviation (NRMSD), which provides an average percent error over time. Results from the head impact tests indicate that the proposed 6aω scheme is capable of producing angular accelerations and linear accelerations transformed to a remote location that are comparable to that determined from the NAP scheme in both low and high speed impact conditions. The 3aω scheme was found to be unable to provide accurate angular accelerations or linear accelerations transformed to a remote location in the high speed head impact condition due to the required numerical differentiation. Both the 6aω and 3aω schemes were capable of measuring accurate angular displacement while the NAP instrumentation was unable to produce accurate angular displacement due to double numerical integration. The proposed 6aω scheme appears to be capable of measuring accurate 6 DOF kinematics of the head in any severity of impact conditions.     

Protein aggregation and amyloidosis: confusion of the kinds?

Recent years have witnessed major advances in our understanding of the structural basis of protein aggregation on several fronts. Firstly, high-resolution structural information that remained elusive for many years was provided by a series of studies of amyloid fibers using NMR, X-ray crystallography and electron microscopy, thereby confirming earlier models based on lower resolution observations. Secondly, studies of the sequence determinants of protein aggregation culminated in the development of computer algorithms that predict aggregation-prone sequences with good accuracy, allowing the design of mutations that reduce aggregation. Thirdly, based on the first results from such predictions and on statistical analysis of naturally occurring aggregating sequences, a picture is emerging in which aggregation-prone sequences are capped by gatekeeper residues that oppose aggregation. In addition to their aggregation-opposing function, it seems that gatekeeper residues are also important in determining chaperone selectivity for strongly aggregating regions. Finally, recent computational and experimental work shows that preventing aggregation does not necessarily mean that amyloid formation is prevented and vice versa. Thus, although aggregation and amyloidosis correlate to a certain extent, they are different processes and should be treated as such.     

AMP-Activated Protein Kinase: A Universal Regulator of Autophagy?

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests that AMPK inhibits mTORC1 and autophagy also in non-starved cells. Various Ca²⁺ mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate AMPK via activation of Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β), and this pathway is required for Ca²⁺-induced mTORC1 inhibition and autophagy. Thus, we propose that an increase in free cytosolic Ca²⁺ ([Ca²⁺]c) induces autophagy via the CaMKK/β-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected.

Addendum to:

Control of Macroautophagy by Calcium, Calmodulin-Dependent Kinase Kinase-β and Bcl-2

M. Høyer-Hansen, L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, I.S. Mathiasen and M. Jäättelä

Mol Cell 2007; 25:193-205     

Protein particulates: another generic form of protein aggregation?

Protein aggregation is a problem with a multitude of consequences, ranging from affecting protein expression to its implication in many diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid fibrils, structures associated with diseases such as Alzheimer's disease. The ability to form amyloid fibrils is now regarded as a property generic to all polypeptide chains. Here we show that around the isoelectric point a different generic form of aggregation can also occur by studying seven widely different, nonrelated proteins that are also all known to form amyloid fibrils. Under these conditions gels consisting of relatively monodisperse spherical particulates are formed. Although these gels have been described before for beta-lactoglobulin, our results suggest that the formation of particulates in the regime where charge on the molecules is minimal is a common property of all proteins. Because the proteins used here also form amyloid fibrils, we further propose that protein misfolding into clearly defined aggregates is a generic process whose outcome depends solely on the general properties of the state the protein is in when aggregation occurs, rather than the specific amino acid sequence. Thus under conditions of high net charge, amyloid fibrils form, whereas under conditions of low net charge, particulates form. This observation furthermore suggests that the rules of soft matter physics apply to these systems.     

Lipopolysaccharide-Binding Protein for Monitoring of Postoperative Sepsis: Complemental to C-Reactive Protein or Redundant?

Introduction

To prospectively evaluate the performance of Lipopolysaccharide-Binding Protein (LBP) in prediction of hospital mortality and its correlation to C-reactive Protein (CRP), we studied sixty consecutive, postoperative patients with sepsis admitted to the university hospital intensive care unit.

Measurements and Methods

Plasma LBP and CRP were serially measured from day(d)1 (onset of sepsis) to d14 in parallel with clinical data until d28. Predictive value and correlation of LBP and CRP were analyzed by Receiver Operating Characteristic (ROC) curve analysis and Pearson''s test, respectively.

Main Results

LBP and CRP showed the highest levels on d2 or d3 after the onset of sepsis with no significant difference between survivors and nonsurvivors. Only at d7, nonsurvivors had significantly (p = .03) higher levels of CRP than survivors. Accordingly, in ROC analysis, concentration of CRP and LBP on d7 poorly discriminated survivors from nonsurvivors (area under curve = .62 and .55, respectively) without significant difference between LBP- and CRP-ROC curves for paired comparison. LBP and CRP plasma levels allocated to quartiles correlated well with each other (r² = .95; p = .02). Likewise, changes in plasma concentrations of LBP and CRP from one observation to the next showed a marked concordance as both parameters concomitantly increased or decreased in 76% of all cases.

Conclusions

During the first 14 days of postoperative sepsis, LBP plasma concentrations showed a time course that was very similar to CRP with a high concordance in the pattern of day-to-day changes. Furthermore, like CRP, LBP does not provide a reliable clue for outcome in this setting.     

Do bacteria differentiate between degrees of nanoscale surface roughness?

Whereas the employment of nanotechnology in electronics and optics engineering is relatively well established, the use of nanostructured materials in medicine and biology is undoubtedly novel. Certain nanoscale surface phenomena are being exploited to promote or prevent the attachment of living cells. However, as yet, it has not been possible to develop methods that completely prevent cells from attaching to solid surfaces, since the mechanisms by which living cells interact with the nanoscale surface characteristics of these substrates are still poorly understood. Recently, novel and advanced surface characterisation techniques have been developed that allow the precise molecular and atomic scale characterisation of both living cells and the solid surfaces to which they attach. Given this additional capability, it may now be possible to define boundaries, or minimum dimensions, at which a surface feature can exert influence over an attaching living organism.This review explores the current research on the interaction of living cells with both native and nanostructured surfaces, and the role that these surface properties play in the different stages of cell attachment.     

Dual-specificity protein kinases: will any hydroxyl do?

Protein kinases are classified by the target amino acid in their substrates. Those protein kinases that phosphorylate hydroxyamino acids comprise two groups, the protein-tyrosine and protein-serine/threonine kinases, which, until recently, had been thought to be mutually exclusive. However, several new protein kinases have been discovered that, by the criterion of primary structure, would be classified as protein-serine/threonine kinases but which, surprisingly, are able to phosphorylate tyrosine residues. Even more surprising, there are reports of protein kinases that are capable of phosphorylating both tyrosine and serine/threonine residues. We review and discuss recent developments concerning these 'dal-specificity' protein kinases.     

Cyclin-dependent kinases: a new cell cycle motif?

Increasing evidence suggests that the eukaryotic cell cycle is controlled at several checkpoints by different members of a novel class of protein kinase, the cyclin-dependent kinases. To phosphorylate their substrates, these enzymes bind to proteins of the cyclin family--proteins that are synthesized and degraded at specific points in each cell cycle. The most well known of these kinases is the 34 kDa product of the cdc2 gene in fission yeast, p34cdc2; however, several putative cyclin-dependent kinases have now been cloned or identified. Some of these closely resemble p34cdc2. Here we review these new proteins, their potential roles in the cell cycle and the cyclins with which they may interact.