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issn:1742-464
1.  BioV Suite - A collection of programs for the study of transport protein evolution 
The FEBS journal  2012;279(11):2036-2046.
The Bio-V Suite is a collection of python scripts designed specifically for bioinformatic research regarding transport protein evolution. The Bio-V Suite contains nine powerful programs for Unix-based environments, each of which can be run as a standalone tool or be accessed in a programmatic fashion. These programs and their functions are as follows: TMStats generates topological statistics for transport proteins. GSAT performs shuffle-based binary alignments and is fully scalable. It can cross compare two FASTA files or individual sequences. Protocol1 performs remote PSI-BLAST searches and filters redundant/similar sequences and annotates them. Protocol2 finds homologues between FASTA lists and generates graphical reports. TSSearch uses a rapid search algorithm to find distant homologues in FASTA files in a heuristic manner. SSearch is the exhuastive version of TSSearch. GBlast will identify potential transport proteins in any genome/proteome file, or find similar transport protein homologues between two different genomes/proteomes before generating a graphical report. AncientRep will find putative transmembrane repeat units using a list of homologues. DefineFamily will generate a FASTA list to represent an entire TC family. These nine programs are tabulated with descriptions of their capabilities in Table 1.
doi:10.1111/j.1742-4658.2012.08590.x
PMCID: PMC3978091  PMID: 22568782
transport proteins; protein evolution; transport classification database; homology; TMS repeats
2.  Engineering active siRNA therapeutics 
The FEBS journal  2010;277(23):4805.
PMCID: PMC3971426  PMID: 21125720
3.  Identification of the heparin-binding domains of the interferon-induced protein kinase, PKR 
The FEBS journal  2005;272(6):1425-1439.
PKR is an interferon-induced serine-threonine protein kinase that plays an important role in the mediation of the antiviral and antiproliferative actions of interferons. PKR is present at low basal levels in cells and its expression is induced at the transcriptional level by interferons. PKR’s kinase activity stays latent until it binds to its activator. In the case of virally infected cells, double-stranded (ds) RNA serves as PKR’s activator. The dsRNA binds to PKR via two copies of an evolutionarily conserved motif, thus inducing a conformational change, unmasking the ATP-binding site and leading to autophosphorylation of PKR. Activated PKR then phosphorylates the α-subunit of the protein synthesis initiation factor 2 (eIF2α) thereby inducing a general block in the initiation of protein synthesis. In addition to dsRNA, polyanionic agents such as heparin can also activate PKR. In contrast to dsRNA-induced activation of PKR, heparin-dependent PKR activation has so far remained uncharacterized. In order to understand the mechanism of heparin-induced PKR activation, we have mapped the heparin-binding domains of PKR. Our results indicate that PKR has two heparin-binding domains that are nonoverlapping with its dsRNA-binding domains. Although both these domains can function independently of each other, they function cooperatively when present together. Point mutations created within these domains rendered PKR defective in heparin-binding, thereby confirming their essential role. In addition, these mutants were defective in kinase activity as determined by both in vitro and in vivo assays.
doi:10.1111/j.1742-4658.2005.04575.x
PMCID: PMC3969814  PMID: 15752359
domain mapping; dsRNA; heparin; interferon; protein kinase
4.  Insights into the suppressor of T-cell receptor (TCR) signaling-1 (Sts-1)-mediated regulation of TCR signaling through the use of novel substrate-trapping Sts-1 phosphatase variants 
The FEBS journal  2013;281(3):696-707.
High affinity substrate-trapping protein tyrosine phosphatases have been widely used both to investigate the endogenous targets of many phosphatases and to address questions of substrate specificity. Herein, we extend the concept of a substrate-trapping phosphatase to include an enzyme of the histidine phosphatase superfamily. This is the first description of substrate-trapping technology applied to a member of the histidine phosphatase family. The phosphatase suppressor of T-cell receptor signaling (Sts)-1 has recently been reported to negatively regulate signaling downstream of the T-cell receptor. We generated high-affinity substrate-trapping variants of Sts-1 by mutagenesis of key active site residues within the phosphatase catalytic domain. Mutation of both the nucleophilic His380 and the general acid Glu490 yielded Sts-1 enzymes that were catalytically inactive but showed high affinity for an important tyrosine kinase in T cells that Sts-1 is known to regulate, Zap-70. Sts-1 substrate-trapping mutants isolated tyrosine-phosphorylated Zap-70 from lysates of activated T cells, validating Zap-70 as a possible substrate for Sts-1 and highlighting the efficacy of the mutants as substrate-trapping agents. Inhibition of the Zap-70 interaction by vanadate suggests that the substrate-trapping effect occurred via the Sts-1 phosphatase active site. Finally, overexpression of Sts-1 substrate-trapping mutants in T cells blocked T-cell receptor signaling, confirming the inhibitory effect of Sts-1 on Zap-70.
doi:10.1111/febs.12615
PMCID: PMC3968691  PMID: 24256567
histidine phosphatase superfamily; suppressor of T-cell receptor signaling (Sts) proteins; T-cell receptor (TCR) signaling
5.  Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacy 
The FEBS journal  2009;276(22):6576-6585.
To enhance silencing and avoid off-target effects, siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5′ end is less stably hybridized relative to the end containing the passenger strand 5′ end. One means by which this is accomplished is to introduce a terminal mismatch, typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5′ end. However, there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here, the silencing efficiency of siRNAs with a terminal mismatch generated either by altering the guide strand (at the 5′ end, nucleotide 1) or the passenger strand (nucleotide 19 from the 5′ end) was examined. Subsequently, we studied the relationship between the silencing efficiency of the siRNAs and their binding to the RNA-induced silencing complex loading complex proteins HIV transactivating response RNA-binding protein and Dicer in H1299 cytoplasmic extracts. Binding of siRNA and the transactivating response RNA-binding protein was significantly reduced by terminal mismatches, which largely agrees with the reduction in eventual silencing efficacy of the siRNAs. Single terminal mismatches led to a small increase in Dicer binding, as expected, but this did not lead to an improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always improve target silencing, and that care should be taken when designing siRNAs using this technique.
doi:10.1111/j.1742-4658.2009.07364.x
PMCID: PMC3967519  PMID: 19811537
Dicer; mismatches; RNA interference; short interfering RNA; TRBP
6.  Unfolding dynamics of the mucin SEA domain probed by force spectroscopy suggest that it acts as a cell protective device 
The FEBS journal  2013;280(6):10.1111/febs.12144.
MUC1 and other membrane-associated mucins harbor long, up to a micrometer, extended highly glycosylated mucin domains and SEA domains situated on their extracellular parts. These mucins line luminal tracts and organs, and are anchored to the apical cell membrane by a transmembrane domain. The SEA domain is a highly conserved domain that undergoes a molecular strain-dependent autocatalytic cleavage during folding in the endoplasmic reticulum, a process required for apical plasma membrane expression. So far no specific function has been designated for the SEA domain. Here, we constructed a recombinant protein consisting of three SEA domains in tandem and used force spectroscopy to assess the dissociation force required to unfold individual, folded SEA domains. Force-distance curves revealed three peaks, each representing unfolding of a single SEA domain. Fitting the observed unfolding events to a worm-like chain model yielded an average contour length of 32 nm per SEA domain. Analysis of forces applied on the recombinant protein revealed an average unfolding force of 168 pN for each SEA domain at a loading rate of 25 nNs−1. Thus, the SEA domain may act as a breaking point that can dissociate before the plasma membrane is breached when mechanical forces are applied to cell surfaces.
doi:10.1111/febs.12144
PMCID: PMC3746175  PMID: 23331320
Atomic Force Microscopy; AFM; Single-Molecule Force Spectroscopy; MUC1; SEA domain; Mucin
7.  Role of α-synuclein penetration into the membrane in the mechanisms of oligomer pore formation 
The FEBS journal  2012;279(6):1000-1013.
Parkinson’s disease (PD) and Dementia with Lewy bodies are common disorders of the aging population characterized by the progressive accumulation of α-synuclein (α-syn) in the CNS. Aggregation of α-syn into oligomers with a ring-like appearance has been proposed a role in toxicity. However, the molecular mechanisms and the potential sequence of events involved in the formation of pore-like structures are unclear. We utilized computer modeling and cell-based studies to investigate the process of α-syn (wild type and A53T) oligomerization in membranes. The studies suggest that α-syn rapidly penetrates the membrane, changing its conformation from α-helical toward a coiled structure. This penetration facilitate the incorporation of additional α-syn monomers to the complex, and subsequent displacement of phospholipids, and formation of oligomers in the membrane. This process occurred more rapidly, and with more favorable energy of interaction for mutant A53T compared with wild type α-syn. After 4 ns of simulation for the protein-membrane model α-syn penetrated through two thirds of the membrane. By 9 ns, the penetration of the annular α-syn oligomers can result in the formation of pore-like structures that fully perforate the lipid bilayer. Experimental incubation of recombinant α-syn in synthetic membranes resulted in the formation of similar pore-like complexes. Moreover, mutant (A53T) α-syn had a greater tendency to accumulate in neuronal membrane fractions in cell cultures, resulting in greater neuronal permeability with the calcein efflux assay. These studies provide a sequential molecular explanation for the process of α-syn oligomerization in the membrane, and support the role of formation of pore-like structures in the pathogenesis of the neurodegenerative process in PD.
doi:10.1111/j.1742-4658.2012.08489.x
PMCID: PMC3925782  PMID: 22251432
8.  Investigation by site-directed mutagenesis of the role of cytochrome P450 2B4 non-active site residues in protein-ligand interactions based on crystal structures of the ligand-bound enzyme 
The FEBS journal  2011;279(9):1607-1620.
SUMMARY
Residues located outside of the active site of cytochromes P450 2B have exhibited importance in ligand binding, structural stability, and drug metabolism. However, contributions of non-active site residues to the plasticity of these enzymes are not known. Thus, a systematic investigation was undertaken of unique residue-residue interactions found in crystal structures of P450 2B4 in complex with 4-(4-chlorophenyl)imidazole (4-CPI), a closed conformation, or in complex with bifonazole, an expanded conformation. Nineteen mutants distributed over eleven sites were constructed, expressed in E. coli, and purified. Most mutants showed significantly decreased expression, especially in the case of interactions found in the 4-CPI structure. Six mutants (H172A, H172F, H172Q, L437A, E474D, and E474Q) were chosen for detailed functional analysis. Among these, the Ks of H172F for bifonazole was ~20-times higher than wild type 2B4, and the Ks of L437A for 4-CPI was ~50-times higher than wild type, leading to significantly altered inhibitor selectivity. Enzyme function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), and 7-benzyloxyresorufin (7-BR). H172F was inactive with all three substrates, and L437A did not turn over 7-BR. Furthermore, H172A, H172Q, E474D and E474Q showed large changes in kcat/KM for each of the three substrates, in some cases up to 50-fold. Concurrent molecular dynamics simulations yield distances between some of the residues in these putative interaction pairs that are not consistent with contact. The results indicate that small changes in the protein scaffold lead to large differences in solution behavior and enzyme function.
doi:10.1111/j.1742-4658.2011.08411.x
PMCID: PMC3911823  PMID: 22051155
cytochrome P450; CYP2B; polymorphism; site-directed mutagenesis
9.  GENETIC MANIPULATION OF MYOBLASTS AND A NOVEL PRIMARY MYOSATELLITE CELL CULTURE SYSTEM: COMPARING AND OPTIMIZING APPROACHES 
The FEBS journal  2013;280(3):827-839.
The genetic manipulation of skeletal muscle cells in vitro is notoriously difficult, especially when using undifferentiated muscle cell lines (myoblasts) or primary muscle stem cells (myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 cells and in a novel system, primary rainbow trout myosatellite cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 cells after optimizing cell density and reagent:DNA ratio, although GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing cell density, although only 30% of cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of cells were transduced with only 106 viral genomes (vg) compared to 54% with 108 vg AAV2 and 23% with 109 vg BAC. Lentivirus also transduced 90% of primary trout myosatellites compared to 1–10% with AAV2 and BAC. The phosphoglycerate kinase 1 promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 cells and both were effective in trout myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle cell systems and could therefore help overcome a common technological barrier.
doi:10.1111/febs.12072
PMCID: PMC3558597  PMID: 23173931
muscle; myoblast; myosatellite; transfection; transduction
10.  Role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of Dr+ Escherichia coli receptor protein Decay Accelerating Factor (DAF or CD55) by Nitric oxide 
The FEBS journal  2013;280(3):840-854.
We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr+). The epithelial invasion of Dr+ E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by down-regulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5′-untranslated region and mapped NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5′-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3′-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. The NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF.
doi:10.1111/febs.12073
PMCID: PMC3558599  PMID: 23176121
Nitric oxide; Decay Accelerating Factor; mRNA stability; Sp1; S-nitrosylation
11.  Exercise Training-Induced Adaptations Associated with Increases in Skeletal Muscle Glycogen Content 
The FEBS journal  2013;280(3):916-926.
Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism for increased muscle glycogen, we studied the effects of exercise training on glycogen regulatory proteins in rat skeletal muscle. Female Sprague Dawley rats performed voluntary wheel running for 1, 4, or 7 weeks. After 7 weeks of training, insulin-stimulated glucose uptake was increased in epitrochlearis muscle. Compared to sedentary control rats, muscle glycogen did not change after 1 week of training, but increased significantly after 4 and 7 weeks. The increases in muscle glycogen were accompanied by elevated glycogen synthase activity and protein expression. To assess the regulation of glycogen synthase, we examined its major activator, protein phosphatase 1 (PP1), and its major deactivator, glycogen synthase kinase 3 (GSK3). Consistent with glycogen synthase activity, PP1 activity was unchanged after 1 week of training but significantly increased after 4 and 7 weeks of training. Protein expression of RGL(GM), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1, GSK3 phosphorylation did not follow the pattern of glycogen synthase activity. The ~40% decrease in GSK-3α phosphorylation after 1 week of exercise training persisted until 7 weeks and may function as a negative feedback to elevated glycogen. Our findings suggest that exercise training-induced increases in muscle glycogen content could be regulated by multiple mechanisms including enhanced insulin sensitivity, glycogen synthase expression, allosteric activation of glycogen synthase and PP1activity.
doi:10.1111/febs.12085
PMCID: PMC3558638  PMID: 23206309
exercise; skeletal muscle; glycogen synthase; GSK-3; rat
12.  Self-trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro 
The FEBS journal  2013;280(4):1084-1094.
The opportunistic pathogen Pseudomonas aeruginosa ranks among leading causes of nosocomial infections. The type III secretion system (T3SS) aids acute P. aeruginosa infections by injecting potent cytotoxins into host cells to suppress the host's innate immune response. Expression of all T3SS-related genes is strictly dependent upon the transcription factor ExsA. Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest. The presented work focuses on the ExsA-ExsC-ExsD-ExsE signaling cascade that ties host cell contact to the up-regulation of T3SS gene expression. Prior to T3SS induction, the anti-activator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions. Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC resulting in the release of ExsA and an up-regulation of the T3SS. Previous studies have shown that the ExsD-ExsA interactions are not freely reversible. Because independently folded ExsD and ExsA were not found to interact, it has been hypothesized that folding intermediates of the two proteins form the complex. Here we demonstrate for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription in vitro and that no other cellular factors are required. More significantly, we show that independently folded ExsD and ExsA are capable of interacting, but only at 37°C and not at 30°C. Guided by the crystal structure of ExsD, we designed a monomeric variant of the protein and demonstrate that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30°C. We propose that this unique mechanism plays an important role in T3SS regulation.
doi:10.1111/febs.12103
PMCID: PMC3621117  PMID: 23279839
ExsA; ExsD; Pseudomonas aeruginosa; thermoregulation; type III secretion
13.  Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms 
The FEBS journal  2013;280(3):880-891.
Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, and the expression and developmental regulation of myofilament proteins in mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues with a lacZ reporter gene driven by cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in a precise synchrony with that in the heart. Nonetheless, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically preset developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart.
doi:10.1111/febs.12076
PMCID: PMC3734956  PMID: 23176202
cardiac muscle; myofilament protein; troponin isoform switch; development
14.  Protein Tyrosine Phosphatases: From Housekeeping Enzymes to Master-Regulators of Signal Transduction 
The FEBS journal  2013;280(2):346-378.
There are many misconceptions surrounding the roles of protein phosphatases in the regulation of signal transduction, perhaps the most damaging of which is the erroneous view that these enzymes exert their effects merely as constitutively active housekeeping enzymes. On the contrary, the phosphatases are critical, specific regulators of signaling in their own right and serve an essential function, in a coordinated manner with the kinases, to determine the response to a physiological stimulus. This review is a personal perspective on the development of our understanding of the protein tyrosine phosphatase (PTP) family of enzymes. I have discussed various aspects of the structure, regulation and function of the PTP family, which I hope will illustrate the fundamental importance of these enzymes to the control of signal transduction.
doi:10.1111/febs.12077
PMCID: PMC3662559  PMID: 23176256
15.  Development of the diaphragm, a skeletal muscle essential for mammalian respiration 
The FEBS journal  2013;280(17):10.1111/febs.12274.
The mammalian diaphragm muscle is essential for respiration, and thus it is among the most critical of the skeletal muscles in the human body. Defects in diaphragm development, leading to congenital diaphragmatic hernias (CDH), are common birth defects and result in severe morbidity or mortality. Given its functional importance and the frequency of congenital defects, an understanding of diaphragm development normally and during herniation is important. We review the current knowledge of the embryological origins of the diaphragm, diaphragm development and morphogenesis, and the genetic and developmental etiology of diaphragm birth defects.
doi:10.1111/febs.12274
PMCID: PMC3879042  PMID: 23586979
diaphragm; Congenital Diaphragmatic Hernia; CDH; muscle; development; tendon
16.  Structural Basis for Protein Phosphatase 1 Regulation and Specificity 
The FEBS journal  2012;280(2):596-611.
The ubiquitous Ser/Thr Protein Phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (≥200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity.
doi:10.1111/j.1742-4658.2012.08509.x
PMCID: PMC3350600  PMID: 22284538
protein phosphatase 1; enzyme regulation; enzyme specificity; structural biology
17.  Signalling by protein phosphatases and drug development: a systems-centred view 
The FEBS journal  2012;280(2):751-765.
Protein-modification cycles catalysed by opposing enzymes, such as kinases and phosphatases form the backbone of signalling networks. Whereas historically, kinases have been at the research forefront, a systems-centred approach reveals predominant roles of phosphatases in controlling the network response times and the spatiotemporal profiles of signalling activities. Emerging evidence suggests that phosphatase kinetics are critical for the network function and cell-fate decisions. Protein phosphatases can operate as both immediate and delayed regulators of signal transduction, capable of attenuating or amplifying signalling. This versatility of phosphatase action emphasises the need for systems biology approaches to comprehend cellular signalling networks and predict the cellular outcomes of combinatorial drug interventions.
doi:10.1111/j.1742-4658.2012.08522.x
PMCID: PMC3368988  PMID: 22340367
phosphatases; spatiotemporal dynamics; signalling cascades; systems biology; drug discovery
18.  Laforin, a protein with many faces: glucan phosphatase, adapter protein, et alii 
The FEBS journal  2012;280(2):525-537.
SUMMARY
Lafora disease (LD) is a rare, fatal neurodegenerative disorder characterized by the accumulation of glycogen-like inclusions in the cytoplasm of cells from most tissues of affected patients. 100 years since the first description of these inclusions, the molecular bases underlying the processes involved in LD physiopathology are finally being elucidated. The main cause for the disease relies on the activity of two proteins, the dual specificity phosphatase laforin and the E3-ubiquitin ligase malin, that form a functional complex. Laforin is unique in humans since it is comprised of a carbohydrate binding module attached to a cysteine-based catalytic dual specificity phosphatase domain. Laforin directly dephosphorylates glycogen, but other proteinaceous substrates, if existent, have remained elusive. Recently, an emerging set of laforin binding partners apart from malin have been described, suggestive of laforin roles unrelated to its catalytic activity. Further investigations based on different transgenic mice models have shown that the laforin-malin complex is also involved in other cellular processes such as response to ER stress and misfolded proteins clearance by the lysosomal pathway. However, controversial data and some missing links still make difficult to assess the concrete relationship between glycogen deregulation and neuronal damage leading to the fatal symptoms observed in LD patients, such as myoclonic seizures and epilepsy. Consequently, clinical treatments are far from being achieved. In the present review, we focus on the knowledge of laforin biology not only as a glucan phosphatase, but also as an adaptor protein involved in several physiological pathways.
doi:10.1111/j.1742-4658.2012.08549.x
PMCID: PMC3371293  PMID: 22364389
Laforin; malin; glucan phosphatase; Lafora disease; Lafora bodies; glycogen; autophagy; ER stress
19.  Methods to monitor classical protein-tyrosine phosphatase oxidation 
The FEBS journal  2012;280(2):459-475.
SUMMARY
Reactive oxygen species (ROS), particularly H2O2, act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pKa. This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling.
doi:10.1111/j.1742-4658.2012.08626.x
PMCID: PMC3439553  PMID: 22577968
Protein-tyrosine phosphatases; Tyrosyl phosphorylation; Signal transduction; Oxidation; Reactive oxygen species; Activity-based probes; Mass spectrometry; Dimedone
20.  Small molecule tools for functional interrogation of protein tyrosine phosphatases 
The FEBS journal  2012;280(2):731-750.
The importance of protein tyrosine phosphatases (PTPs) in the regulation of cellular signaling is well established. Malfunction of PTP activity is also known to be associated with cancer, metabolic syndromes, autoimmune disorders, neurodegenerative and infectious diseases. However, a detailed understanding of the roles played by the PTPs in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific small molecule agents. In addition, the therapeutic benefits of modulating this target class are underexplored due to lack of suitable chemical probes. Potent and specific PTP inhibitors could significantly facilitate functional analysis of the PTPs in complex cellular signal transduction pathways and may constitute valuable therapeutics in the treatment of several human diseases. We will highlight the current challenges and opportunities in developing PTP-specific small molecule agents. We will also review available selective small molecule inhibitors developed for a number of PTPs, including PTP1B, TC-PTP, SHP2, Lyp, HePTP, CD45, PTPβ, PTPγ, PTPRO, VHR, MKP-1, MKP-3, Cdc25, YopH, mPTPA, and mPTPB.
doi:10.1111/j.1742-4658.2012.08718.x
PMCID: PMC3495087  PMID: 22816879
Tyrosine phosphorylation; protein tyrosine phosphatases; small molecule inhibitors; chemical probes; potency and specificity; high throughput screening; structure-based design; virtual screening; fragment-based focus library
21.  Cryo-electron microscopy: A primer for the non-microscopist 
The FEBS journal  2012;280(1):28-45.
Cryo-electron microscopy (cryo-EM) is increasingly becoming a mainstream technology for studying the architecture of cells, viruses and protein assemblies at molecular resolution. Recent developments in microscope design and imaging hardware, paired with enhanced image processing and automation capabilities, appear poised to further advance the effectiveness of cryo-EM methods. These developments promise to increase the speed and extent of automation and to improve the resolutions that can be achieved, rendering this technology capable of determining a wide variety of biological structures. Additionally, established modalities for structure determination, such as X-ray crystallography and nuclear magnetic resonance spectroscopy, are being routinely integrated with cryo-EM density maps to achieve atomic-resolution models of complex, dynamic molecular assemblies. In this review, which is directed towards readers who are not experts in cryo-EM methodology, we provide an overview of emerging themes in the application of this technology to the investigation of diverse questions in biology and medicine. We discuss the ways in which these methods are being used to study structures of macromolecular assemblies that range in size from whole cells to small proteins. Finally, we include a description of how the structural information obtained by cryo-EM is deposited and archived in a publicly accessible database.
doi:10.1111/febs.12078
PMCID: PMC3537914  PMID: 23181775
22.  N-terminal phosphorylation of PP2A/Bβ2 regulates translocation to mitochondria, Drp1 dephosphorylation, and neuronal survival 
The FEBS journal  2012;280(2):662-673.
Summary
The neuron-specific Bβ2 regulatory subunit of protein phosphatase 2A (PP2A), a product of the spinocerebellar ataxia type 12 disease gene PPP2R2B, recruits heterotrimeric PP2A to the outer mitochondrial membrane (OMM) through its N-terminal mitochondrial targeting sequence. OMM-localized PP2A/Bβ2 induces mitochondrial fragmentation, thereby increasing susceptibility to neuronal insults. Here, we report that PP2A/Bβ2 activates the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) by dephosphorylating Ser656, a highly conserved inhibitory phosphorylation site targeted by the neuroprotective PKA/AKAP1 kinase complex. We further show that translocation of PP2A/Bβ2 to mitochondria is regulated by phosphorylation of Bβ2 at three N-terminal Ser residues. Phosphomimetic substitution of Ser20-22 renders Bβ2 cytosolic, blocks Drp1 dephosphorylation and mitochondrial fragmentation, and abolishes the ability of Bβ2 overexpression to induce apoptosis in cultured hippocampal neurons. Ala substitution of Ser20-22 to prevent phosphorylation has the opposite effect, promoting association of Bβ2 with mitochondria, Drp1 dephosphorylation, mitochondrial fission, and neuronal death. OMM translocation of Bβ2 can be attenuated by mutation of residues in close proximity to the catalytic site, but only if Ser20-22 are available for phosphorylation, suggesting that PP2A/Bβ2 autodephosphorylation is necessary for OMM association, likely by uncovering the net positive charge of the mitochondrial targeting sequence. These results reveal another layer of complexity in the regulation of the mitochondrial fission/fusion equilibrium and its physiological and pathophysiological consequences in the nervous system.
doi:10.1111/j.1742-4658.2012.08631.x
PMCID: PMC3549015  PMID: 22583914
protein phosphatase 2A; neuronal survival; dynamin-related protein1; mitochondrial fission; protein phosphorylation
23.  Structural characterization of the second intradiscal loop of the photoreceptor tetraspanin RDS 
The FEBS journal  2012;280(1):127-138.
Vertebrate photoreceptors contain a unique tetraspanin protein known as retinal degeneration slow (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases and more than 70% of these mutations are located in the second intradiscal (D2) loop, highlighting the importance of this region. Here we examined conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with RDS’ non-glycosylated homologue ROM-1. The RDS D2 loop was expressed in E. coli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP-D2, was purified to homogeneity. Circular dichroism (CD) spectroscopy showed that the wild-type (WT) D2 loop consists of ~21% α-helix, ~20% β-sheet and ~59% random coil. D2 loop fusion proteins carrying disease-causing mutations in RDS (e.g R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to WT D2). In particular, C150S, C214S, and N244H showed significant reductions in the α-helicity. Nevertheless, the thermal stability of the mutants was unchanged compared to WT, and all mutants were all capable of interacting with ROM-1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I-TASSER model of the RDS D2 loop predicted structure consistent with the CD experiments and with the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS-associated retinal degeneration.
doi:10.1111/febs.12055
PMCID: PMC3652632  PMID: 23121719
RDS; tetraspanin; CD spectroscopy; macular dystrophy; retinitis pigmentosa
24.  Receptor-type tyrosine phosphatases ligands: looking for the needle in the haystack 
The FEBS journal  2012;280(2):388-400.
Reversible protein phosphorylation plays a pivotal role in intercellular communication. Together with protein tyrosine kinases, protein tyrosine phosphatases (PTPs) are involved in the regulation of key cellular processes by controlling the phosphorylation levels of diverse effectors. Among PTPs, receptor-like protein tyrosine phosphatases (RPTPs) are involved in important developmental processes, particularly in the formation of the nervous system. Until recently, few ligands had been identified for RPTPs, making it difficult to grasp the effects these receptors have on cellular processes as well as the mechanisms through which their functions are mediated. However, several potential RPTP ligands have now been identified to provide us with unparalleled insights into RPTP function. In this review, we will focus on the nature and biological outcomes of these extracellular interactions between RPTPs and their associated ligands.
doi:10.1111/j.1742-4658.2012.08653.x
PMCID: PMC3753797  PMID: 22682003
Phosphatases; Ligands; Receptor-ligand interactions; RPTP
25.  Suppression of Survival Signalling Pathways by the Phosphatase PHLPP 
The FEBS journal  2012;280(2):572-583.
Summary
The recently discovered PH (pleckstrin homology) domain Leucine rich repeat Protein Phosphatase (PHLPP) family is emerging as a central component in suppressing cell survival pathways. Originally discovered in a rational search for a phosphatase that directly dephosphorylates and inactivates Akt, PHLPP is now known to potently suppress cell survival both by inhibiting proliferative pathways and by promoting apoptotic pathways. In the first instance, PHLPP directly dephosphorylates a conserved regulatory site (termed the hydrophobic motif) on Akt, protein kinase C (PKC), and S6 kinase, thereby terminating signalling by these pro-survival kinases. In the second instance, PHLPP dephosphorylates and thus activates the pro-apoptotic kinase Mst1, thereby promoting apoptosis. PHLPP is deleted in a large number of cancers and the genetic deletion of one isozyme in a PTEN (phosphatase and tensin homolog located on chromosome 1) +/− prostate cancer model results in increased tumourigenesis, underscoring the role of PHLPP as a tumour suppressor. This review summarises the targets and cellular actions of PHLPP, with emphasis on its role as a tumour suppressor in the oncogenic PI3K (phosphoinositide 3-kinase)/Akt signalling cascade.
doi:10.1111/j.1742-4658.2012.08537.x
PMCID: PMC3770140  PMID: 22340730
PHLPP; Akt; PKC; PI3K; mTOR; PTEN; phosphatase; prostate cancer; chronic lymphocytic leukaemia

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