Biochemical Analysis of Murine Wnt Proteins Reveals both Shared and Distinct Properties

The murine Wnt family of proteins consists of at least 12 members that possess significant amino acid homology. Current evidence suggests that these proteins are secreted cell-signaling molecules which are likely to have multiple roles during both embryonic development and oncogenesis. Although the biochemical properties of Wnt-1 have been thoroughly examined, less is known about the characteristics of other Wnt family members. We have compared the properties of six murine Wnt proteins (Wnt-1, Wnt-3a, Wnt-5a, Wnt-5b, Wnt-6, and Wnt-7b) transiently expressed in COS cells. All members enter the endoplasmic reticulum (ER) and are glycosylated. However, all six Wnt proteins are primarily retained in the ER in association with BiP, a resident ER protein that binds to improperly folded proteins and prevents their secretion and/or promotes proper folding. Although all Wnt family members examined are similarly processed, one notable difference was identified. Whereas addition of suramin to COS cell cultures significantly increases the levels of all six Wnts in the medium, the addition of heparin only influences the levels of Wnt-1, Wnt-6, and Wnt-7b.     

Molecular Characterization of Cryptosporidium molnari Reveals a Distinct Piscine Clade

Multilocus phylogenetic analysis of small-subunit (SSU) rRNA and actin from Cryptosporidium molnari clustered this species with the C. molnari-like genotype of an isolate from the guppy, although the two fish isolates seem to be distinct species. The analysis of available piscine genotypes provides some support for cladistic congruence of the genus Piscicryptosporidium, but additional piscine genotypes are needed.Recent reviews accept more than 20 valid cryptosporidium species (7, 20), and characterization of additional isolates is expanding this list rapidly (http://www.vetsci.usyd.edu.au/staff/JanSlapeta/icrypto/index.htm). In addition, numerous morphotypes or genotypes have been proposed whose taxonomic affiliation is unsettled due to incomplete characterization according to minimum consensus standards (5, 7, 24). Five species have been proposed for fish isolates (15), but only Cryptosporidium molnari and Cryptosporidium scophthalmi (2, 4) stand as valid species (20), although not without discussion (7). Fish cryptosporidia present some unique features, which have even led to the genus Piscicryptosporidium being proposed (13). However, lack of genetic support keeps this genus and several fish morphotypes as incertae sedis (12, 15, 24). Detailed biological data on C. molnari and C. scophthalmi have been previously presented (3, 18, 19), but no molecular characterization has yet been conducted, thus hampering species identification of other fish isolates (7, 24) and evaluation of their relationships within the genus (15). Ribosomal and actin gene data on an isolate from guppy fish (Poecilia reticulata) have been obtained, and preliminary analyses of these sequences indicated a basal position in the cryptosporidial tree (17). Although it was regarded as C. molnari-like, biological characterization of this isolate was limited. The purpose of this work was to provide the necessary C. molnari comparative genetic data and to clarify the relationship of available fish isolates in a phylogenetic context.     

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development ^{1, 2}. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease^{3, 4, 5}. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination^{6, 7} is visualized in Xenopus ectodermal cells to study Wnt signal transduction⁸. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion ^{8, 9}. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.Download video file.^{(43M, mov)}     

Quantitative Proteomics Reveals a Dynamic Association of Proteins to Detergent-resistant Membranes upon Elicitor Signaling in Tobacco

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used ¹⁴N/¹⁵N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in the regulation of the signaling process may be conserved between plant and animals.The plasma membrane of eukaryotes delineates the interface between the cell and the environment. Thus it is particularly involved in environmental signal recognition and their transduction into intracellular responses, playing a crucial role in many essential functions such as cell nutrition (involving transport of solutes in and out of the cell) or response to environmental modifications (including defense against pathogens).Over the last 10 years, a new aspect of the plasma membrane organization has arisen from biophysical and biochemical studies performed with animal cells. Evidence has been given that the various types of lipids forming this membrane are not uniformly distributed inside the bilayer but rather spatially organized (1). This leads in particular to the formation of specialized phase domains, also called lipid rafts (2, 3). Recently a consensus emerged on the characteristics of these domains. Both proteins and lipids contribute to the formation and the stability of membrane domains that should be called “membrane rafts” and are envisaged as small (10–200-nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes (4). Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions (5). Because of their particular lipidic composition (enrichment in sterol, sphingolipids, and saturated fatty acids), these domains form a liquid ordered phase inside the membrane. This structural characteristic renders them resistant to solubilization by non-ionic detergents, and this property has been widely used to isolate lipid rafts as detergent-resistant membranes (DRMs)¹ for further analysis (1). The most important hypothesis to explain the function of these domains is that they provide for lateral compartmentalization of membrane proteins and thereby create a dynamic scaffold to organize certain cellular processes (5). This ability to temporally and spatially organize protein complexes while excluding others conceivably allows for efficiency and specificity of cellular responses. In yeasts and animal cells, the association of particular proteins with these specialized microdomains has emerged as an important regulator of crucial physiological processes such as signal transduction, polarized secretion, cytoskeletal organization, generation of cell polarity, and entry of infectious organisms in living cells (6). Much of the early evidence for a functional role of lipid rafts came from studies of hematopoietic cells in which multichain immune receptors including the high affinity IgE receptor (FcεRI), the T cell receptor, and the B cell receptor (BCR) translocate to lipid rafts upon cross-linking (7). Moreover this signaling involves the relocalization of several proteins; for instance the ligation of the B cell antigen receptor with antigen induced a dissociation of the adaptor protein ezrin from lipid rafts (8). This release of ezrin acts as a critical trigger that regulates lipid raft dynamics during BCR signaling.In plants, the investigations of the presence of such microdomains are very recent and limited to a reduced number of publications (for a review, see Ref. 9). A few years ago, Peskan et al. (10) reported for the first time the isolation of Triton X-100-insoluble fractions from tobacco plasma membrane. Mongrand et al. (11) provided a detailed analysis of the lipidic composition of such a detergent-resistant fraction indicating that it was highly enriched in a particular species of sphingolipid (glycosylceramide) and in several phytosterols (stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol) compared with the whole plasma membrane from which it originates. Similar results were then obtained with DRMs prepared from Arabidopsis thaliana cell cultures (12) and from Medicago truncatula roots (13). So the presence in plant plasma membrane of domains sharing with animal rafts a particular lipidic composition, namely strong enrichment in sphingolipids together with free sterols and sterol conjugates, the latter being specific to the plant kingdom (11, 13), now seems established. In plant only a few evidences suggest in vivo the role of dynamic clustering of plasma membrane proteins, and they refer to plant-pathogen interaction. A cell biology study reported the pathogen-triggered focal accumulation of components of the plant defense pathway in the plasma membrane (PM), a process reminiscent of lipid rafts (14). Consistently a proteomics study of tobacco DRMs led to the identification of 145 proteins among which a high proportion were linked to signaling in response to biotic stress, cellular trafficking, and cell wall metabolism (15). This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in such physiological functions.Cryptogein belongs to a family of low molecular weight proteins secreted by many species of the oomycete Phytophthora named elicitins that induce a hypersensitivity-like response and an acquired resistance in tobacco (16). To understand molecular processes triggered by cryptogein, its effects on tobacco cell suspensions have been studied for several years. Early events following cryptogein treatment include fixation of a sterol molecule (17, 18); binding of the elicitor to a high affinity site located on the plasma membrane (19); alkalinization of the extracellular medium (20); efflux of potassium, chloride, and nitrate (20, 21); fast influx of calcium (22); mitogen-activated protein kinases activation (23, 24); nitric oxide production (25, 26); and development of an oxidative burst (27, 28). We previously identified NtrbohD, an NADPH oxidase located on the plasma membrane, as responsible for the reactive oxygen species (ROS) production occurring a few minutes after challenging tobacco Bright Yellow 2 (BY-2) cells with cryptogein (29). The fact that most of these very early events involve proteins located on the plasma membrane and that one of them, NtrbohD, has been demonstrated as exclusively associated to DRMs in a sterol-dependent manner (30) prompted us to analyze the modifications of DRM proteome after cryptogein treatment. In the present study, we aimed to confirm the hypothesis that, as observed in animal cells, the dynamic association to or exclusion of proteins from lipid rafts could participate in the signaling process occurring during biotic stress in plants.To achieve this goal, we had to set up a quantitative assay allowing a precise comparison of the amounts of each protein in DRMs extracted from either control or cells treated with cryptogein. Among several technologies, we excluded DIGE (31), recently used to analyze whole cell proteome variations in plants (32–34), because membrane proteins are poorly soluble in the detergents used for two-dimensional electrophoresis; this limitation is all the more marked for proteins selected on the basis of their insolubility in non-ionic detergent, the criteria for DRMs isolation. Stable isotope labeling of proteins or peptides combined with MS analysis represents alternative strategies for accurate, relative quantification of proteins on a global scale (35, 36). In these approaches, proteins or peptides of two different samples are differentially labeled with stable isotopes, combined in an equal ratio, and then jointly processed for subsequent MS analysis. Relative quantification of proteins is based on the comparison of signal intensities or peak areas of isotope-coded peptide pairs extracted from the respective mass spectra. Stable isotopes can be introduced either chemically into proteins/peptides via derivatization of distinct functional groups of amino acids or metabolically during protein biosynthesis (for a review, see Ref. 37). Metabolic labeling strategies are based on the in vivo incorporation of stable isotopes during growth of organisms. Nutrients or amino acids in a defined medium are replaced by their isotopically labeled (¹⁵N, ¹³C, or ²H) counterparts eventually resulting in uniform labeling of proteins during the processes of cell growth and protein turnover (38). As a consequence, differentially labeled cells or organisms can be combined directly after harvesting. This minimizes experimental variations due to separate sample handling and thus allows a relative protein quantification of high accuracy.¹⁴N/¹⁵N labeling has been recently proved to be suitable for comparative experiments performed with whole plants (39–42) and in plant suspension cells where the level of incorporation is equal to the isotopic purity of the salt precursor (43, 44). It has been used successfully to analyze some variations induced in A. thaliana plasma membrane proteome following heat shock (45) or cadmium exposure (46) and to compare phosphorylation levels of plasma membrane proteins after challenge of Arabidopsis cells with elicitors of defense reaction (47). In the present study, we used a mineral ¹⁴N/¹⁵N metabolic labeling of tobacco BY-2 cells before treatment with cryptogein and subsequent isolation of DRMs. The DRM proteins were further analyzed by one-dimensional SDS-PAGE and digested by trypsin, and peptides were subjected to microcapillary high performance LC-MS/MS (nano-LC-MS/MS). This metabolic method allowed a complete labeling of the proteome, and consequently a major drawback of this method is probably the difficulty to perform an exhaustive analysis of the very large amount of data generated. To solve this problem, a new quantification module of the MFPaQ software (48) was developed, allowing the automatic quantification of the identified peptides. The results derived from the program were validated through a comparison with manual quantification. Thus, we achieved the complete analysis of the DRM proteome variation and identified four proteins whose abundance in DRMs was decreased and one that was enriched in DRMs upon elicitation. The biological relevance of these results, which indicate that, in plant as in animals, the dynamic association of proteins to membrane domains is part of a signaling pathway, will be further discussed.     

Molecular Analysis of Human and Canine Staphylococcus aureus Strains Reveals Distinct Extended-Host-Spectrum Genotypes Independent of Their Methicillin Resistance

Staphylococcus aureus causes a wide range of infectious diseases in humans and various animal species. Although presumptive host-specific factors have been reported, certain genetic lineages seem to lack specific host tropism, infecting a broad range of hosts. Such Extended-Host-Spectrum Genotypes (EHSGs) have been described in canine infections, caused by common regional human methicillin-resistant S. aureus (MRSA) lineages. However, information is scarce about the occurrence of methicillin-susceptible S. aureus (MSSA) EHSGs. To gain deeper insight into EHSG MSSA and EHSG MRSA of human and canine origin, a comparative molecular study was carried out, including a convenience sample of 120 current S. aureus (70 MRSA and 50 MSSA) isolates obtained from infected dogs. spa typing revealed 48 different spa types belonging to 16 different multilocus sequence typing clonal complexes (MLST-CCs). Based on these results, we further compared a subset of canine (n = 48) and human (n = 14) strains, including isolates of clonal complexes CC5, CC22, CC8, CC398, CC15, CC45, and CC30 by macrorestriction (pulsed-field gel electrophoresis [PFGE]) and DNA-microarray analysis. None of the methods employed was able to differentiate between clusters of human and canine strains independently of their methicillin resistance. In contrast, DNA-microarray analysis revealed 79% of the 48 canine isolates as carriers of the bacteriophage-encoded human-specific immune evasion cluster (IEC). In conclusion, the high degree of similarity between human and canine S. aureus strains regardless of whether they are MRSA or MSSA envisions the existence of common genetic traits that enable these strains as EHSGs, challenging the concept of resistance-driven spillover of MRSA.     

Distinct Modes of Inhibition by Sclerostin on Bone Morphogenetic Protein and Wnt Signaling Pathways

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.     

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.     

Stage-Specific Regulation of Reprogramming to Induced Pluripotent Stem Cells by Wnt Signaling and T Cell Factor Proteins

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Direct Comparison of Mononucleated and Binucleated Cardiomyocytes Reveals Molecular Mechanisms Underlying Distinct Proliferative Competencies

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Crystal Structure of Human Seryl-tRNA Synthetase and Ser-SA Complex Reveals a Molecular Lever Specific to Higher Eukaryotes

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Proteomic Analysis of Pure Human Airway Gland Mucus Reveals a Large Component of Protective Proteins

Airway submucosal glands contribute to innate immunity and protect the lungs by secreting mucus, which is required for mucociliary clearance and which also contains antimicrobial, anti-inflammatory, anti-proteolytic and anti-oxidant proteins. We stimulated glands in tracheal trimmings from three lung donors and collected droplets of uncontaminated mucus as they formed at the gland orifices under an oil layer. We analyzed the mucus using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis identified 5486 peptides and 441 proteins from across the 3 samples (269–319 proteins per subject). We focused on 269 proteins common to at least 2 0f 3 subjects, of which 102 (38%) had protective or innate immunity functions. While many of these have long been known to play such roles, for many others their cellular protective functions have only recently been appreciated in addition to their well-studied biologic functions (e.g. annexins, apolipoproteins, gelsolin, hemoglobin, histones, keratins, and lumican). A minority of the identified proteins are known to be secreted via conventional exocytosis, suggesting that glandular secretion occurs via multiple mechanisms. Two of the observed protective proteins, major vault protein and prohibitin, have not been observed in fluid from human epithelial cultures or in fluid from nasal or bronchoalveolar lavage. Further proteomic analysis of pure gland mucus may help clarify how healthy airways maintain a sterile environment.     

Molecular Cloning of Echis ocellatus Disintegrins Reveals Non-Venom-Secreted Proteins and a Pathway for the Evolution of Ocellatusin

We report the cloning and sequence analysis of Echis ocellatus cDNAs coding for dimeric disintegrin subunits and for the short disintegrin ocellatusin. All the dimeric disintegrin subunit messengers belong to the short-coding class, indicating that short messengers may be more widely distributed than previously thought. Mass spectrometric analysis of the HPLC-separated venom proteins was performed to characterize the dimeric disintegrins expressed in the venom proteome. In addition to previously reported EO4 and EO5 heterodimers, a novel dimeric disintegrin containing RGD- and KGD-bearing subunits was identified. However, a WGD-containing polypeptide encoded by clone Eo1-1 was not detected in the venom, suggesting the occurrence of larger genomic than proteomic diversity, which could represent part of a non-venom-secreted reservoir of disintegrin that may eventually acquire physiological relevance for the snake upon changes of ecological niches and prey habits. On the other hand, the realization of the existence of two distinct messengers coding for the short disintegrin ocellatusin reveals key events of the evolutionary emergence of the short disintegrin ocellatusin from a short-coding dimeric disintegrin precursor by two nucleotide mutations. [Reviewing Editor: Dr. Bryan Grieg Fry]     

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients’ sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.     

Methionine Mistranslation Bypasses the Restraint of the Genetic Code to Generate Mutant Proteins with Distinct Activities

Although mistranslation is commonly believed to be deleterious, recent evidence indicates that mistranslation can be actively regulated and be beneficial in stress response. Methionine mistranslation in mammalian cells is regulated by reactive oxygen species where cells deliberately alter the proteome through incorporating Met at non-Met positions to enhance oxidative stress response. However, it was not known whether specific, mistranslated mutant proteins have distinct activities from the wild-type protein whose sequence is restrained by the genetic code. Here, we show that Met mistranslation with and without Ca²⁺ overload generates specific mutant Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) proteins substituting non-Met with Met at multiple locations. Compared to the genetically encoded wild-type CaMKII, specific mutant CaMKIIs can have distinct activation profiles, intracellular localization and enhanced phenotypes. Our results demonstrate that Met-mistranslation, or “Met-scan” can indeed generate mutant proteins in cells that expand the activity profile of the wild-type protein, and provide a molecular mechanism for the role of regulated mistranslation.     

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

VPS4 proteins are AAA⁺ ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA⁺ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His₆-VPS4A on liposomes containing Ni²⁺-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.     

Searching for Local Similarities between HIV-1 and Human Proteins. Application to Vaccines

A method for identification of fragments with high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions whose fragments are frequent in human proteins, and regions exhibiting high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV infection either through induction of cross-reacting effectors of the immune system or through molecular mimicry of physiologically important human proteins, leading to alteration of homeostasis of the organism, is discussed. Most of the regions identified in HIV-1 proteins contain either T-cell (CD8⁺ CTL or CD4⁺ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines that would allow exclusion of epitopes with (immuno)pathogenic potential are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions whose fragments are frequent in human proteins, as well as of regions exhibiting pronounced local similarity to proteins that provide for important physiological functions.     

Wnt/Wingless Pathway Activation and Chromosome 6 Loss Characterise a Distinct Molecular Sub-Group of Medulloblastomas Associated with a Favourable Prognosis

The accurate assessment of disease risk remains a major goal in children with medulloblastoma. Activation of the canonical Wnt/Wingless (Wnt/Wg) signalling pathway occurs in up to 25% of cases and is associated with a favourable disease outcome. To explore the molecular pathogenesis of Wnt/Wg-active medulloblastomas, and to investigate any genetic basis for their observed clinical behaviour, we assessed a series of primary medulloblastomas for evidence of Wnt/Wg pathway activation, alongside a genome-wide analysis of associated copy-number aberrations. Cases displaying evidence of Wnt/Wg activation (CTNNB1 mutation and/or β-catenin nuclear stabilisation) were exclusively associated with a distinct genomic signature involving loss of an entire copy of chromosome 6 but few other aberrations (p