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BBA - Proteins and Proteomics (v.1784, #1)

Editorial Board (pp. ii).

Special issue: Inhibitors of protein kinases by David Shugar (pp. 1-2).

Mammalian NDR protein kinases: From regulation to a role in centrosome duplication by Alexander Hergovich; Hauke Cornils; Brian A. Hemmings (pp. 3-15).

The NDR (nuclear Dbf2-related) family of kinases is highly conserved from yeast to human, and has been classified as a subgroup of the AGC group of protein kinases based on the sequence of the catalytic domain. Like all other members of the AGC class of protein kinases, NDR kinases require the phosphorylation of conserved Ser/Thr residues for activation. Importantly, NDR family members have two unique stretches of primary sequence: an N-terminal regulatory (NTR) domain and an insert of several residues between subdomains VII and VIII of the kinase domain. The kinase domain insert functions as an auto-inhibitory sequence (AIS), while binding of the co-activator MOB (Mps-one binder) proteins to the NTR domain releases NDR kinases from inhibition of autophosphorylation. However, despite such advances in our understanding of the molecular activation mechanism(s) and physiological functions of NDR kinases in yeast and invertebrates, most biological NDR substrates still remain to be identified. Nevertheless, by showing that the centrosomal subpopulation of human NDR1/2 is required for proper centrosome duplication, the first biological role of human NDR1/2 kinases has been defined recently. How far NDR-driven centrosome overduplication could actually contribute to cellular transformation will also be discussed.

Keywords: Abbreviations; AGC; PKA/PKG/PKC-like; NDR; nuclear Dbf2-related; MOB; Mps-one binder; LATS; large tumour suppressor; Trc; tricornered; SAX-1; sensory axon guidance-1; PP2A; protein phosphatase type 2A; MST; Mammalian Ste20-like kinase; SWH; Salvador/Warts/Hippo; Hpo; Hippo; Sav; Salvador; Mer; Merlin; Ex; Expanded; Ft; Fat; Yki; Yorkie; RASSF1A; RAS-association domain family protein 1A; PLK4; Polo-like kinase 4; Cdk2; cyclin-dependent kinase 2; NTR; N-terminal regulatory; AIS; auto-inhibitory sequence; SPB; spindle pole bodies (SPB); MEN; mitotic exit network; SIN; septation initiation networkNDR/LATS protein kinase; Activation mechanism; MOB binding; Subcellular localization; Centriole duplication; Tumour suppressor; Cellular transformation

Signaling through cAMP and cAMP-dependent protein kinase: Diverse strategies for drug design by Susan S. Taylor; Choel Kim; Cecilia Y. Cheng; Simon H.J. Brown; Jian Wu; Natarajan Kannan (pp. 16-26).

The catalytic subunit of cAMP-dependent protein kinase has served as a prototype for the protein kinase superfamily for many years while structures of the cAMP-bound regulatory subunits have defined the conserved cyclic nucleotide binding (CNB) motif. It is only structures of the holoenzymes, however, that enable us to appreciate the molecular features of inhibition by the regulatory subunits as well as activation by cAMP. These structures reveal for the first time the remarkable malleability of the regulatory subunits and the CNB domains. At the same time, they allow us to appreciate that the catalytic subunit is not only a catalyst but also a scaffold that mediates a wide variety of protein:protein interactions. The holoenzyme structures also provide a new paradigm for designing isoform-specific activators and inhibitors of PKA. In addition to binding to the catalytic subunits, the regulatory subunits also use their N-terminal dimerization/docking domain to bind with high affinity to A Kinase Anchoring Proteins using an amphipathic helical motif. This targeting mechanism, which localizes PKA near to its protein substrates, is also a target for therapeutic intervention of PKA signaling.

Keywords: Abbrevations; cAMP; cyclic-3′,5′-adenosine monophosphate; PKA; cAMP-dependent protein kinase; PKI; heat stable protein kinase A inhibitor; PDK1; phosphoinositide-dependent kinase-1; ATP; adenosine triphosphate; D/D domain; dimerization/docking domain; AKIP1; A Kinase Interacting Protein 1; H/DMS; Hydrogen/deuterium exchange coupled with mass spectrometry; PBC; phosphate binding cassette; CNB domain; cyclic nucleotide binding domain; C-subunit; catalytic subunit; R-subunit; regulatory subunit; N-lobe; amino-terminal lobe; C-lobe; carboxy-terminal; N-tail; N-terminal tail; C-tail; C-terminal tail; CLT; C-lobe Tether; NLT; N-lobe Tether; AST; Active Site Tether; CLA; C-lobe Anchor; CBD; cyclic nucleotide binding; AKAP; A Kinase Anchoring Protein; D-AKAP2; dual specific AKAP2; PIP3; Phospho-inositide triphosphate; Akt/Pkb; Protein Kinase B; H/D exchange; hydrogen/deuterium exchange; CAP; catabolite gene activator protein; AGS kinase; PKA PKG PKC and related kinases; PKC; Protein Kinase C; LiReC; ligand-regulated competition; HTS; high throughput screenPKA; PKI; Holoenzyme; RIα; RIIα; Signaling; Allostery; Dynamic; Kinase inhibitor; A Kinase Anchoring Proteins (AKAPs)

Analogous regulatory sites within the αC-β4 loop regions of ZAP-70 tyrosine kinase and AGC kinases by Natarajan Kannan; Andrew F. Neuwald; Susan S. Taylor (pp. 27-32).

The precise positioning of the flexible C-helix in the catalytic core is a critical step in the activation of most protein kinases. Consequently, the αC-β4 loop, which anchors the C-helix to the catalytic core, is highly conserved and mediates key structural interactions that serve as a hinge for C-helix movement. While these hinge interactions are conserved across diverse eukaryotic protein kinase structures, some families such as AGC kinases diverge from the canonical hinge interactions. This divergence was recently proposed to facilitate an alternative mode of regulation wherein a conserved C-terminal tail interacts with the αC-β4 loop to position the C-helix. Here we show how interactions between the αC-β4 loop and the N-terminal SH2 domain of ZAP-70 tyrosine kinase are mechanistically and functionally analogous to interactions between the αC-β4 loop and the C-terminal tail of AGC kinases. Such cis regulation of protein kinase activity may be a feature of other eukaryotic protein kinase families as well.

Keywords: Abbreviations; AGC kinases; PKA, PKG, PKC and related kinases; ZAP-70 tyrosine kinase; Zeta-chain-associated protein kinase 70; Syk; Spleen tyrosine kinase; MEK1; Map kinase kinase-1; ErbB2; Erythroblastic leukemia viral oncogene homolog 2; SH2; Src homology domain 2; PH; Pleckstrin homology domain; Cdk2; cyclin-dependent kinase 2; CMGC; Cyclin-dependent kinases mitogen activated protein kinase glycogen synthase kinase-2 and Ck2; EPK; Eukaryotic protein kinase; ELK; Eukaryotic-like kinasesPhosphorylation; Catalysis; Allostery; Genome; Evolution; Inhibitor; Dynamic; Pathway

Too much of a good thing: The role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2 by James S. Duncan; David W. Litchfield (pp. 33-47).

CK2 is a highly conserved protein serine/threonine kinase that is ubiquitously distributed in eukaryotes, constitutively active and has been implicated in multiple cellular functions, as well as in tumorigenesis and transformation. Elevated CK2 activity has been associated with the malignant transformation of several tissues and is associated with aggressive tumor behaviour. While the precise roles of CK2 in tumorigenesis remain incompletely understood, mounting evidence suggests a role for CK2 in the protection of cells from apoptosis via the regulation of tumor suppressor and oncogene activity. Consequently, CK2 has emerged as a potential therapeutic target, and strategies to inhibit CK2 have been ongoing in pre-clinical trials. This review will focus on published evidence highlighting the molecular mechanisms by which CK2 functions in the promotion of tumorigenesis, as well as review current strategies being used to inhibit CK2.

Keywords: Abbreviations; TBB; 4,5,6,7-tetrabromo-1H-benzotriazole; TBBz; 4,5,6,7-tetrabromobenzimidazole; DMAT; 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; TBCA; tetrabromocinnamic acid; IQA; (5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl)acetic acid; Inhibitor 7; 5,6,8-trichloro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid; PTM; post-translational modifications; PML; promyelocytic leukemia gene; PI3′K pathway; phosphatidylinositol-3 kinase pathway; NF-κB; nuclear factor-κB; APC; adenomatous polyposis coli; PTEN; phosphatase and tensin homolog deleted on chromosome 10; MM cells; multiple myeloma cells; AS protein kinase; analog-sensitive protein kinaseSignal transduction; Protein kinase; CK2; Tumorigenesis; Chemical inhibitor; Proteomic

Unique MAP Kinase binding sites by Radha Akella; Thomas M. Moon; Elizabeth J. Goldsmith (pp. 48-55).

Map kinases are drug targets for autoimmune disease, cancer, and apoptosis-related diseases. Drug discovery efforts have developed MAP kinase inhibitors directed toward the ATP binding site and neighboring “DFG-out” site, both of which are targets for inhibitors of other protein kinases. On the other hand, MAP kinases have unique substrate and small molecule binding sites that could serve as inhibition sites. The substrate and processing enzyme D-motif binding site is present in all MAP kinases, and has many features of a good small molecule binding site. Further, the MAP kinase p38α has a binding site near its C-terminus discovered in crystallographic studies. Finally, the MAP kinases ERK2 and p38α have a second substrate binding site, the FXFP binding site that is exposed in active ERK2 and the D-motif peptide induced conformation of MAP kinases. Crystallographic evidence of these latter two binding sites is presented.

Keywords: MAP kinases; Inhibitor binding; FXFP; Sulindac; PD98059; X-ray crystallographyAbbreviations; CD-domain; Common docking domain; D-Motif; Docking motif or docking site in MAP kinase substrates and MAP kinase processing enzymes; DFG-out; unusual conformation in well conserved DFG in subdomain VII; ED; hydrophobic docking groove (that is flanked by D and E residues); ERK; Extracellular response kinase; JNK; c-Jun N-terminal kinase; MAPK; Mitogen activated protein kinase; MAP2K; Mitogen activated protein kinase kinase; MAPKAP kinase; Mitogen activated protein kinase activated protein kinase

Src family kinases: Regulation of their activities, levels and identification of new pathways by Evan Ingley (pp. 56-65).

While the Src family of protein tyrosine kinases (SFK), and the main ancillary molecules involved in their regulation, have been studied for many years, the details of their interplay are not fully understood and thus remain under active investigation. Additionally, new players that coordinate their regulation and direct their signalling cascades are also being uncovered, shedding new light on the complexity of these signalling networks. Through the utilization of novel interaction assays, several new interconnecting mediators that are helping to show the elegance of Src family kinase regulation have been discovered. This review outlines SFK regulation, the discovery of the Csk binding protein (Phosphoprotein Associated with Glycosphingolipid-enriched microdomains, Cbp/PAG), and its role in regulating SFK kinase activity status, as well as protein levels. Further, details of the methods used to identify this dual mode of regulation can be applied to delineate the full gamut of SH2/SH3-directed SFK pathways and, indeed, those of any tyrosine kinase. Using Lyn as a model SFK, we and others have shown that Cbp recruits negative regulators of COOH-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk) after Lyn is activated and bound to Cbp. Lyn phosphorylates Cbp on multiple tyrosine residues, including two that can bind Lyn's SH2 domain with high affinity. Lyn also phosphorylates Y314, which recruits Csk/Ctk to phosphorylate Lyn at its Y508 negative site, allowing an inactive conformation to form. However, the pY508 site has a low affinity for Lyn's SH2 domain, while the Cbp sites have high affinity. Thus, until these Cbp sites are dephosphorylated, Lyn can remain active. Intriguingly, phosphorylated Y314 also binds the suppressor of cytokine signalling 1 (SOCS1), resulting in elevated ubiquitination and degradation of Lyn. Thus, a single phosphotyrosine residue within Cbp co-ordinates a two-phase process involving distinct negative regulatory pathways that allow inactivation, followed by degradation, of SFKs.

Keywords: Abbreviations; Abl; Abelson leukemia oncogene; Akt; Ak transforming oncogene also called; PKB; protein kinase B; Arg; Abl homolog; Blk; B lymphocyte tyrosine kinase; Btk; Bruton's tyrosine kinase; Cbl; Cas-Br-M (murine) oncogene; Cbp; Csk binding protein; CD45; transmembrane receptor-like tyrosine phosphatase; Chk; Csk homologous kinase; Csk; COOH-terminal Src kinase; EBP50; ERM-binding phosphoprotein 50; Etk; endothelial and epithelial tyrosine kinase; Fer; Fes related homolog; Fes; Fujinami poultry sarcoma oncogene; Fgr; Gardner-Rasheed feline sarcoma oncogene; Fyn; Oncogene related to Src and Yes; Hck; Hemopoietic cell kinase; Itk; IL2-inducible T-cell kinase; JAK; Janus kinase; LAT; linker for activation of T cells; Lck; leukocyte-specific protein tyrosine kinase; LIME; Lck-interacting membrane protein; Lyn; Yes homolog; NTAL; Non-T Cell Activation Linker; PAG; phosphoprotein associated with GEMs, also called Cbp; PDZ; PSD95/DLG/ZO-1 homolog domain; PEP; proline-enriched tyrosine phosphatase; PI3-kinase; phosphatidyl inositol-3' kinase; PLCγ; phospholipase C gamma; SFK; Src Family Kinase; SH2; Src homology 2 domain; SH3; Src homology 3 domain; SHP1; tandem SH2 domain-containing protein tyrosine phosphatase; SHP2; SHP1 homolog; SOCS1; suppressor of cytokine signalling 1; Src; cellular homologue of transforming gene of Rous sarcoma virus; STAT; signal transducer and activator of transcription; TCPTP; T-cell protein tyrosine phosphatase; Y2H; yeast two-hybrid; Yes; Yamaguchi sarcoma oncogeneSrc family kinase; C-terminal Src kinase; Csk binding protein; SFK regulation; Phosphotyrosine-specific yeast two-hybrid; SH2 interaction

Sphingosine kinase signalling in immune cells: Potential as novel therapeutic targets by Alirio J. Melendez (pp. 66-75).

During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis.Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca²⁺ mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the potential for targeting SphKs to suppress inflammation and other pathological conditions.

Keywords: Sphingosine kinase; Sphingosine-1-phosphate; Immune cell; Inflammation; Monocytes/macrophage; Neutrophil; Mast cell; Lymphocytes; Signalling

Inhibitors of c-Jun N-terminal kinases—JuNK no more? by Marie A. Bogoyevitch; Peter G. Arthur (pp. 76-93).

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.

Keywords: Abbreviations; CVB3; Coxsackievirus B3; HIV; Human immunodeficiency virus; JNKs; c-Jun N-terminal kinases; MAPKs; mitogen-activated protein kinases; MPTP; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PARP-1; poly(ADP-ribose) polymerase-1; PDB; Protein DataBase; ROS; Reactive oxygen species; RNAi; RNA interferencec-Jun N-terminal Kinase; NS3, Non-structural protein 3; SP600125; Small molecule ATP-competitive inhibitors; JNK inhibitory peptides; Ischemia/reperfusion damage; Viral infection

Peptide-based fluorescent sensors of protein kinase activity: Design and applications by Vyas Sharma; Qunzhao Wang; David S. Lawrence (pp. 94-99).

Protein kinases control the flow of information through cell-signaling pathways. A detailed analysis of their behavior enhances our ability to understand normal cellular states and to devise therapeutic interventions for diseases. The design and application of “Environmentally-Sensitive”, “Deep-Quench” and “Self-Reporting” sensor systems for studying protein kinase activity are described. These sensors allow real-time activity measurements in a continuous manner for a wide variety of kinases. As these sensors can be adapted from an in vitro screen to imaging kinase activity in living cells, they support both preliminary and later stages of drug discovery.

Keywords: Signal transduction; Sensor; Fluorescent peptide; Assay; Inhibitor; Drug discovery

Histidine phosphorylation in biological systems by Jennifer Puttick; Edward N. Baker; Louis T.J. Delbaere (pp. 100-105).

Histidine phosphorylation is important in prokaryotes and occurs to the extent of 6% of total phosphorylation in eukaryotes. Nevertheless phosphohistidine residues are not normally observed in proteins due to rapid hydrolysis of the phosphoryl group under acidic conditions. Many rapid processes employ phosphohistidines, including the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), the bacterial two-component systems and reactions catalyzed by enzymes such as nucleoside diphosphate kinase and succinyl–CoA synthetase. In the PTS, the NMR structure of the phosphohistidine moiety of the phosphohistidine-containing protein was determined but no X-ray structures of phosphohistidine forms of PTS proteins have been elucidated. There have been crystal structures of a few phosphohistidine-containing proteins determined: nucleoside diphosphate kinase, succinyl–CoA synthetase, a cofactor-dependent phosphoglycerate mutase and the protein PAE2307 from the hyperthermophilic archaeon Pyrobaculum aerophilum. A common theme for these stable phosphohistidines is the occurrence of ion-pair hydrogen bonds (salt bridges) involving the non-phosphorylated nitrogen atom of the histidine imidazole ring with an acidic amino acid side chain.

Keywords: Phosphohistidine; Nucleoside diphosphate kinase; Succinyl–CoA synthetase; Phosphotransfer; PTS; Two-component system

Sperm activation: Role of reactive oxygen species and kinases by Eve de Lamirande; Cristián O’Flaherty (pp. 106-115).

Reactive oxygen species (ROS), such as the superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂^{) and nitric oxide (NO}), when generated at low and controlled levels, act as second messengers. ROS regulate sperm capacitation, which is the complex series of changes allowing spermatozoa to bind to the zona pellucida surrounding the oocyte, induce the acrosome reaction (exocytotic event by which proteolytic enzymes are released) and fertilize the oocyte. Capacitating spermatozoa produce controlled amounts of ROS that regulate downstream events: first, the increase in cAMP, protein kinase A (PKA) activation and phosphorylation of PKA substrates (arginine-X-X-serine/threonine motif; 15–30 min); second, the phosphorylation of MEK (extracellular signal regulated kinase [ERK] kinase)-like proteins (30–60 min) and then that of the threonine–glutamate–tyrosine motif (>1 h); finally, the late tyrosine phosphorylation of fibrous sheath proteins (>2 h). Although all these events are ROS-dependent, the regulation by various kinases, protein kinase C, PKA, protein tyrosine kinases, the ERK pathway, etc. is different. ROS also regulate the acquisition of hyperactivated motility and the acrosome reaction by spermatozoa. ROS action is probably mediated via the sulfhydryl/disulfide pair on sperm proteins. Redundancy, cross talk, and multiple systems acting in parallel point to an array of safeguards assuring the timely function of spermatozoa.

Keywords: Abbreviations; AKAPS; A-kinase anchoring proteins; Arg; arginine; Cys; cysteine; ERK; extracellular signal regulated kinase; Glu; glutamate; H; ₂; O; ₂; hydrogen peroxide; LPC; lysophosphatidylcholine; MEK; ERK kinase; MPM2; monoclonal anti-P-Ser/Thr-Pro antibody; ^NO; nitric oxide; NOS; nitric oxide synthase; O; ₂; ⁻; superoxide anion; PI3K; phosphoinositide-3-kinase; PKA; protein kinase A; PKC; protein kinase C; P-MEK; phospho-MEK; P-PKA; phospho-PKA; P-Tyr; phospho-Tyr; Pro; proline; PTK; protein tyrosine kinase; ROS; reactive oxygen species; Ser; serine; SOD; superoxide dismutase; Thr; threonine; Tyr; tyrosineSpermatozoa; Capacitation; Reactive oxygen species; Protein phosphorylation; Signal transduction

Role of mTOR in physiology and pathology of the nervous system by Lukasz Swiech; Malgorzata Perycz; Anna Malik; Jacek Jaworski (pp. 116-132).

Mammalian target of rapamycin (mTOR) is a serine–threonine protein kinase that regulates several intracellular processes in response to extracellular signals, nutrient availability, energy status of the cell and stress. mTOR regulates survival, differentiation and development of neurons. Axon growth and navigation, dendritic arborization, as well as synaptogenesis, depend on proper mTOR activity. In adult brain mTOR is crucial for synaptic plasticity, learning and memory formation, and brain control of food uptake. Recent studies reveal that mTOR activity is modified in various pathologic states of the nervous system, including brain tumors, tuberous sclerosis, cortical displasia and neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. This review presents current knowledge about the role of mTOR in the physiology and pathology of the nervous system, with special focus on molecular targets acting downstream of mTOR that potentially contribute to neuronal development, plasticity and neuropathology.

Keywords: Abbreviations; Aβ; β-amyloid; AD; Alzheimer's disease; BDNF; brain-derived neurotrophic factor; HD; Huntington's disease; CD; cortical displasia; 4E-BP1; eIF-4E binding protein; KO; knockout; NF1; type I neurofibromatosis; p70S6K; p70 ribosomal S6 kinase; PD; Parkinson's disease; PI3K; phosphoinositide-3′ kinase; PRAS40; proline-rich AKT substrate 40 kDa; Raptor; regulatory-associated protein of mTOR; Rheb; Ras homolog enriched in brain; Rictor; rapamycin-insensitive companion of mTOR; RNAi; RNA interference; SIN1; mammalian stress-activated protein kinase interacting protein 1; mTOR; mammalian target of rapamycin; TSC; tuberous sclerosis; TSC1; tuberous sclerosis complex protein 1, hamartin; TSC2; tuberous sclerosis complex protein 2, tuberin; TSC1/2; tuberous sclerosis complexmTOR; Nervous system; Rapamycin; Neuronal development; Synaptic plasticity; Neurodegenerative disorders

Molecular structure and regulation of phototropin kinase by blue light by Satoru Tokutomi; Daisuke Matsuoka; Kazunori Zikihara (pp. 133-142).

Phototropin (phot) is a blue light photoreceptor in plants that mediates phototropism, chloroplast movement, stomata opening and leaf expansion. The phot molecule has two photoreceptive domains, LOV 1 and 2, in the N-terminal half and the C-terminal half forms Ser/Thr kinase. Phot acts as a blue light-regulated protein kinase. Each LOV domain binds a FMN and undergoes a unique cyclic reaction upon blue light absorption that induces conformational changes in the protein moiety and leads to regulation of the kinase activity, in which LOV2 plays a predominant role in the switching and LOV1 acts to attenuate the light sensitivity. Phot kinase is classified into the AGC kinase group since the consensus amino acid residues and the motifs are well conserved except for the lack of the hydrophobic motif and the presence of additional amino acid sequence in the activation loop. Secondary structure prediction and 3D structure simulation show a α/β fold of the phot kinase similar to that of the catalytic subunit of PKA. The additional sequence forms an extra helix and loops. Docking simulation of the LOV2 domain with phot kinase provided useful information regarding the molecular mechanism underlying the photoregulation of phot kinase.

Keywords: Abbreviations; AGC kinase; cAMP-dependent protein kinase A, cGMP-dependent protein kinase G and phospholipid dependent protein kinase C; A-loop; activation loop; ATP; adenosine triphosphate; cAMP; cyclic-3′, 5′-adenosine monophosphate; C-loop; catalytic loop; FMN; flavin mononucleotide; HM; hydrophobic motif; LOV; Light, Oxygen and Voltage-sensing; MAPK; Mitogen-activated protein kinase; neo1; neochrome1; NPH3; non phototropic hycopotyl3; PAS; Per-ARNT-Sim; phot1; phototropin1; phot2; phototropin 2; PKA; cAMP-dependent protein kinase A; PKD; protein kinase domain; PKI; heat stable protein kinase A inhibitor; P-loops; phosphate-binding loop; PKS1; phytochrome kinase substrate1; PP1; type 1 protein phosphatase; SAXS; small-angle X-ray scatteringPhototropin; LOV; PAS; PKA; PKI; Blue light; Signaling

Evaluation of 4,5,6,7-tetrahalogeno-1 H-isoindole-1,3(2 H)-diones as inhibitors of human protein kinase CK2 by A.G. Golub; O. Ya. Yakovenko; A.O. Prykhod'ko; S.S. Lukashov; V.G. Bdzhola; S.M. Yarmoluk (pp. 143-149).

Protein kinase CK2 (Casein Kinase 2) is an extremely pleiotropic Ser/Thr kinase with high constitutive activity. The observation of CK2 deregulations in various pathological processes suggests that CK2 inhibitors may have a therapeutic value, particularly as anti-neoplastic and antiviral drugs. Here, we present the 4,5,6,7-tetrahalogeno-1 H-isoindole-1,3(2 H)-diones as a novel potent class of CK2 inhibitors. We identified this class of inhibitors by high-throughput docking of a compound collection in the ATP-binding site of human CK2. The most active compounds are 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2 H-isoindol-2-yl)propanoic acid and 2-(4,5,6,7-tetraiodo-1,3-dioxo-1,3-dihydro-2 H-isoindol-2-yl)acetic acid with IC₅₀ values of 0.15 μM and 0.3 μM, respectively. These inhibitors are ATP-competitive and they only minimally inhibit the activities of protein kinases DYRK1a, MSK1, GSK3 and CDK5. Binding modes for the most active inhibitors are proposed.

Keywords: Abbreviations; CK2; protein kinase CK2; TBB; tetrabromobenzotriazole (also known as TBBt); TBBz; tetrabromobenzimidazole; K25; 2-dimethylamino-4,5,6,7-tetrabromo-1; H; -benzimidazole; K37; 2-methylthio-4,5,6,7-tetrabromo-1; H; -benzimidazole; K44; N; ₁; ,; N; ₂; -ethylene-2-methylamino-4,5,6,7-tetrabromo-1; H; -benzimidazole; TID; 4,5,6,7-tetrahalogeno-1; H; -isoindole-1,3(2; H; )-dioneProtein kinase CK2; Inhibitor; 4,5,6,7-tetrahalogeno-1; H; -isoindole-1,3(2; H; )-dione; Docking; Molecular modeling

PI3K/PTEN signaling in tumorigenesis and angiogenesis by Bing-Hua Jiang; Ling-Zhi Liu (pp. 150-158).

The phosphatidyl inositol 3-kinase (PI3K) can be activated by a variety of extracellular signals and involved in a number of cellular processes including cell proliferation, survival, protein synthesis, and tumor growth. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is an antagonist of PI3K. The alterations of PI3K pathway such as activation of oncogenes, gene amplification, and inactivation of tumor suppressors, commonly occur in many human cancers. Angiogenesis is required for tumor growth and metastasis when the tumor reaches more than 1 mm in diameter. Recent studies have shown that PI3K and Akt play an important role in regulating tumor growth and angiogenesis through VEGF and HIF-1 expression. PI3K regulates the expression of these two proteins through HDM2 and p70S6K1 in human cancer cells. The frequent dysregulation of the PI3K/PTEN pathway in human cancer demonstrates that this pathway is an appropriate target for cancer therapeutics. In this review, we describe the recent advances in understanding the PI3K/PTEN pathway, the role and mechanism of PI3K in regulating tumor growth and angiogenesis, and the potential therapeutic opportunities for targeting this pathway for cancer treatment.

Keywords: Abbreviations; PI3K; phosphatidylinositol 3-kinase; RTK; receptor tyrosine kinase; mTOR; mammalian target of rapamycin; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; FGFR; fibroblast growth factor receptor; IGF; insulin-like growth factor; IGF-1R; insulin-like growth factor 1 receptor; VEGF; vascular endothelial growth factor; VEGFR; vascular endothelial growth factor receptor; PH domain; pleckstrin-homology domain; PDK; phosphoinositide-dependent kinase; 4E-BP1; eukaryotic translation initiation factor 4E-binding protein 1; FOXO; forkhead; G6Pase; glucose-6-phosphatase; GSK3; glycogen synthase kinase 3; HDM2; human double minute 2; Cdk4; cyclin-dependent kinase 4; MAPK; mitogen-activated protein kinase; Rb; retinoblastoma protein; cdc25A; cell division cycle 25A; PTEN; phosphatase and tensin homolog deleted on chromosome 10; CDDP; cisplatin; CAM; chicken chorioallantoic membrane; HIF-1; hypoxia-inducible factor 1; PX-316; phosphatidylmyoinositol-1-[(; R; )-2-methoxy-3-octadecyloxyropyl hydrogen phosphate]; API-59-Ome; 9-methoxy-2-methylellipticinium acetate; VQD-002; triciribinePI3K; PTEN; Akt; mTOR; p70S6K1; VEGF; HIF-1; HDM2; Angiogenesis; Tumorigenesis

Targeting phosphoinositide 3-kinase—Moving towards therapy by Romina Marone; Vladimir Cmiljanovic; Bernd Giese; Matthias P. Wymann (pp. 159-185).

Phosphoinositide 3-kinases (PI3K) orchestrate cell responses including mitogenic signaling, cell survival and growth, metabolic control, vesicular trafficking, degranulation, cytoskeletal rearrangement and migration. Deregulation of the PI3K pathway occurs by activating mutations in growth factor receptors or the PIK3CA locus coding for PI3Kα, by loss of function of the lipid phosphatase and tensin homolog deleted in chromosome ten (PTEN/MMAC/TEP1), by the up-regulation of protein kinase B (PKB/Akt), or the impairment of the tuberous sclerosis complex (TSC1/2). All these events are linked to growth and proliferation, and have thus prompted a significant interest in the pharmaceutical targeting of the PI3K pathway in cancer. Genetic targeting of PI3Kγ (p110γ) and PI3Kδ (p110δ) in mice has underlined a central role of these PI3K isoforms in inflammation and allergy, as they modulate chemotaxis of leukocytes and degranulation in mast cells. Proof-of-concept molecules selective for PI3Kγ have already successfully alleviated disease progress in murine models of rheumatoid arthritis and lupus erythematosus. As targeting PI3K moves forward to therapy of chronic, non-fatal disease, safety concerns for PI3K inhibitors increase. Many of the present inhibitor series interfere with target of rapamycin (TOR), DNA-dependent protein kinase (DNA-PK_cs) and activity of the ataxia telangiectasia mutated gene product (ATM). Here we review the current disease-relevant knowledge for isoform-specific PI3K function in the above mentioned diseases, and review the progress of >400 recent patents covering pharmaceutical targeting of PI3K. Currently, several drugs targeting the PI3K pathway have entered clinical trials (phase I) for solid tumors and suppression of tissue damage after myocardial infarction (phases I,II).

Keywords: PI3K; Cancer; Inflammation; Allergy; Pharmacology; Drug development

Insights from site-specific phosphoproteomics in bacteria by Boumediene Soufi; Carsten Jers; Mette Erichsen Hansen; Dina Petranovic; Ivan Mijakovic (pp. 186-192).

Recent advances in mass spectrometry allowed the charting of bacterial serine/threonine/tyrosine phosphoproteomes with unprecedented accuracy, including the acquisition of a large number of phosphorylation sites. Phosphorylated bacterial proteins are involved in some key housekeeping processes, and their phosphorylation is expected to play an important regulatory role. When coupled to stable isotope labeling by amino acids in cell culture (SILAC), high-resolution mass spectrometry allows the detection of changes in the occupancy of phosphorylation sites in response to various stimuli. This and similar approaches promise to lead bacterial phosphoproteomics into the era of systems biology, where the entire phosphorylation-based regulatory networks will be charted, modelled, and ultimately engineered to obtain desired properties.

Keywords: Abbreviations; BY-kinase; Bacterial tyrosine kinase; GPS; group-based phosphorylation scoring; HPr; histidine phosphocarrier protein; MS; mass spectrometry; PTS; phosphoenolpyruvate-dependant carbohydrate phosphotransferase system; SILAC; stable isotope labeling by amino acids in cell cultureBacterial phosphoproteome; Phosphoproteomics; Protein phosphorylation; Kinases; Phosphatases; Phosphopredictor

Mycobacterial Ser/Thr protein kinases and phosphatases: Physiological roles and therapeutic potential by Annemarie Wehenkel; Marco Bellinzoni; Martin Graña; Rosario Duran; Andrea Villarino; Pablo Fernandez; Gwénaëlle Andre-Leroux; Patrick England; Howard Takiff; Carlos Cerveñansky; Stewart T. Cole; Pedro M. Alzari (pp. 193-202).

Reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes, not only in eukaryotes but also in bacteria. A growing body of evidence suggests that Ser/Thr phosphorylation play important roles in the physiology and virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This pathogen uses ‘eukaryotic-like’ Ser/Thr protein kinases and phosphatases not only to regulate many intracellular metabolic processes, but also to interfere with signaling pathways of the infected host cell. Disrupting such processes by means of selective inhibitors may thus provide new pharmaceutical weapons to combat the disease. Here we review the current knowledge on Ser/Thr protein kinases and phosphatases in M. tuberculosis, their regulation mechanisms and putative substrates, and we explore their therapeutic potential as possible targets for the development of new anti-mycobacterial compounds.

Keywords: Abbreviations; STPK; Ser/Thr protein kinase; FHA; forkhead-associated domain; PASTA; penicillin-binding protein and Ser/Thr kinase-associated domain; PTP; protein tyrosine kinase; pThr; phospho-threonine; pSer; phospho-serine; MS; mass spectrometry Mycobacterium tuberculosis; Ser/Thr protein phosphorylation; Protein kinases; Protein phosphatases; Drug design

Conserved herpesvirus protein kinases by Edward Gershburg; Joseph S. Pagano (pp. 203-212).

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however, along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein–Barr virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task.

Keywords: Abbreviations; CHPK; conserved herpesviral protein kinase; HSV; herpes simplex virus; VZV; varicella-zoster virus; HCMV; human cytomegalovirus; EBV; Epstein–Barr virus; KSHV; Kaposi sarcoma associated virus; HHV; human herpesvirus; AIDS; acquired immunodeficiency syndrome; ICP0; infected cell protein 0; SCID; severe combined immunodeficiency; GCV; ganciclovir (2-amino-9-(1,3-dihydroxypropan-2-yloxymethyl)-3H-purin-6-one); OXT-G; oxetanocin G (2-amino-9-[(2R,3R,4R)-3,4-; bis; (hydroxymethyl)oxetan-2-yl]-3H-purin-6-one); ACV; acyclovir (2-amino-9-(2-hydroxyethoxymethyl)-3H-purin-6-one); BV-araU; bravavir (5-[(; E; )-2-bromoethenyl]-1-[(2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl] pyrimidine-2,4-dione); CDV; cidofovir ([(2S)-1-(4-amino-2-oxo-pyrimidin-1-yl)-3-hydroxy-propan-2-yl]oxymethylphosphonic acid); BVDU; brivudin (5-[(; E; )-2-bromoethenyl]-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dione); PCV; penciclovir (2-amino-9-[4-hydroxy-3-(hydroxymethyl)butyl]-3H-purin-6-one); MBV; maribavir ((2S)-2-[5,6-dichloro-2-(propan-2-ylamino)benzoimidazol-1-yl]-5-(hydroxymethyl)oxolane-3,4-diol); NLS; nuclear localization signal; RNAi; RNA interference; MCM; EBNA2; Epstein–Barr nuclear antigen 2; EBNA-LP; Epstein–Barr nuclear antigen leader peptide; GST; glutathione-; S; -transferase; BDCRB; (2R,3R,4S,5R)-2-(2-bromo-5,6-dichloro-benzoimidazol-1-yl)-5-(hydroxymethyl)oxolane-3,4-diol; CDK1; cyclin-dependent kinase 1; JNK; c-Jun N-terminal kinase; MCM; minichromosome maintenance proteinsHerpesvirus; Protein kinase; Serine/Threonine kinase; Protein kinase inhibitor; Antiviral compound

Regulated nucleocytoplasmic trafficking of viral gene products: A therapeutic target? by Gualtiero Alvisi; Stephen M. Rawlinson; Reena Ghildyal; Alessandro Ripalti; David A. Jans (pp. 213-227).

The study of viral proteins and host cell factors that interact with them has represented an invaluable contribution to understanding of the physiology as well as associated pathology of key eukaryotic cell processes such as cell cycle regulation, signal transduction and transformation. Similarly, knowledge of nucleocytoplasmic transport is based largely on pioneering studies performed on viral proteins that enabled the first sequences responsible for the facilitated transport through the nuclear pore to be identified. The study of viral proteins has also enabled the discovery of several nucleocytoplasmic regulatory mechanisms, the best characterized being through phosphorylation. Recent delineation of the mechanisms whereby phosphorylation regulates nuclear import and export of key viral gene products encoded by important human pathogens such as human cytomegalovirus dengue virus and respiratory syncytial virus has implications for the development of antiviral therapeutics. In particular, the development of specific and effective kinase inhibitors makes the idea of blocking viral infection by inhibiting the phosphorylation-dependent regulation of viral gene product nuclear transport a real possibility. Additionally, examination of a chicken anemia virus (CAV) protein able to target selectively into the nucleus of tumor but not normal cells, as specifically regulated by phosphorylation, opens the exciting possibility of cancer cell-specific nuclear targeting. The study of nucleoplasmic transport may thus enable the development not only of new antiviral approaches, but also contribute to anti-cancer strategies.

Keywords: Abbreviations; aa; amino acid; aa; amino acids; ARM; Armadillo; CAV; chicken anemia virus; cdk; cyclin-dependent kinase; CK2; protein kinase CK2; CRM1; chromosome maintenance protein 1; DENV; dengue virus; DF; dengue fever; DHF; dengue hemorrhagic fever; EBV; Epstein–Barr virus; EXP; exportin; HCMV; human cytomegalovirus; HIV; human immunodeficiency virus; hnRNP; heterogenous ribonucleoprotein particle; HSV; herpes simplex virus; IL-8; interleukin-8; IMP; importin; JEV; Japanese encephalitis virus; LMB; leptomycin B; M; matrix; NDV; Newcastle disease virus; NE; nuclear envelope; NES; nuclear export sequence; NLS; nuclear localization signal; NPC; nuclear pore complex; PAP; polymerase accessory protein; PKA; protein kinase A; PKC; protein kinase C; PML; promyelocytic leukaemia protein; PTHrP; parathyroid hormone related protein; Rb; Retinoblastoma protein; RC; replication complex; RdRp; RNA-dependent RNA polymerase; RSV; respiratory syncytial virus; SV40; Simian virus SV40; T-ag; large tumor antigen; TBB; tetrabromobenzotriazole; tNTS; tumor cell-specific nuclear targeting sequence; VP3; viral protein 3; VPs; vesicle packets; vRNA; viral RNA; VSV; vesicular stomatitis virus; VZV; varicella-zoster virus; YFV; yellow fever virusPhosphorylation regulation; Virus; Protein; Nuclear protein import; Nuclear protein export; hCMV ppUL44 processivity factor; SV40 large tumor antigen; dengue virus NS5; Respiratory syncitial virus matrix protein; Chicken anemia viral protein 3; Tumor cell-specific; Protein kinase CK2

The role of cell signaling in poxvirus tropism: The case of the M-T5 host range protein of myxoma virus by Steven J. Werden; Grant McFadden (pp. 228-237).

Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.

Keywords: Abbreviations; ANK; ankyrin; BGMK; baby green monkey kidney; CHO; Chinese hamster ovary; INF; interferon; IRF-3; interferon regulatory factor 3; MNF; myxoma nuclear factor; mTORC; mammalian target of rapamycin complex; MV; myxoma virus; PDK1; 3-phosphoinositide-dependent protein kinase-1; PH; pleckstrin homology; PI-3K; phosphatidylinositol-3-kinase; PIKE-A; phosphatidylinositol-3-kinase enhancer Akt; pMEF; primary mouse embryo fibroblast; RK13; rabbit kidney fibroblast; SCF; Skp, Cullin, F-box; Skp; S-phase kinase-associated protein; STAT1; signal transducer and activator of transcription 1; VARV; variola virus; VSV; vesicular stomatitis virus; VV; vaccinia virusPoxvirus; Virus tropism; Akt; Ankyrin repeat; F-box; Protein kinase

Conserved spatial patterns across the protein kinase family by Lynn F. Ten Eyck; Susan S. Taylor; Alexandr P. Kornev (pp. 238-243).

Protein kinases are a large family of enzymes heavily involved in signal transduction, regulation of metabolism, and control of cell growth and differentiation. These functions require precise recognition of widely diverse signals and substrates, and very detailed control of protein kinase activity. Large molecules interact primarily through recognition of surface features. Comparison of surfaces is complicated by both sequence diversity and conformational variability, including multiple possible rotameric states of side chains. We used a recently developed method of protein surface comparison to compare different serine/threonine and tyrosine kinases. As we have shown, two hydrophobic cores inside a protein kinase molecule are connected by a unique formation, called the “spine”. It exists only in the active conformation of protein kinases and is dynamically disassembled during the inactivation process. Detection of such structures by any other method was not possible as the residues which comprise the spine do not form any sequence or 3D motifs in a traditional sense.

Keywords: Abbreviations; cAMP; Cyclic-3',5' -adenosine monophosphate; PKA; cAMP-dependent protein kinase; ECC; Edge Comparison and Combination; CNB domain; Cyclic Nucleotide Binding domain; PBC; phosphate binding cassette; CAP; catabolite gene activator protein; HCN channel; hyperpolarization-activated, cyclic nucleotide-modulated channel; C-subunit; catalytic subunit of PKA; R-subunit; regulatory subunit of PKA; MAP kinase; mitogen activated protein kinaseProtein kinase; PKA; Regulation; Activation mechanism; Protein surface; Graph-theory method

Flexible ligand–flexible protein docking in protein kinase systems by Chung F. Wong (pp. 244-251).

Quite a few reviews on molecular docking have already appeared. This mini-review focuses on methods that incorporate protein flexibility in docking rather than those that treat protein targets as rigid molecules. This is still a challenging problem but there are encouraging recent advances. These methods will be reviewed particularly in light of their applications to protein kinases and phosphatases. In addition to obtaining correct docking pose, recent developments on exploring docking pathways are also highlighted.

Keywords: Molecular dynamics; Simulated annealing; Mining minima; Docking pathway; Protein phosphatase

Understanding protein folding: Small proteins in silico by Olav Zimmermann; Ulrich H.E. Hansmann (pp. 252-258).

Recent improvements in methodology and increased computer power now allow atomistic computer simulations of protein folding. We briefly review several advanced Monte Carlo algorithms that have contributed to this development. Details of folding simulations of three designed mini proteins are shown. Adding global translations and rotations has allowed us to handle multiple chains and to simulate the aggregation of six beta-amyloid fragments. In a different line of research we have developed several algorithms to predict local features from sequence. In an outlook we sketch how such biasing could extend the application spectrum of Monte Carlo simulations to structure prediction of larger proteins.

Keywords: Protein folding; Aggregation; Generalized-ensemble sampling; Structure prediction

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BBA - Proteins and Proteomics (v.1784, #1)