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In 1956, John G. Kemeny and Paul Oppenheim proposed an approach to intertheoretical reduction as an alternative to that of Ernest Nagel. However, they neglected to provide a clear definition of its basic concept of systematization. After decades of languishing in the shadows, new interest in the KO approach is emerging. Nevertheless, there are still misunderstandings regarding this basic concept. The present paper elucidates this concept by returning to Oppenheim's hitherto little-noticed publications from the 1920s and 1930s, which Kemeny and Oppenheim obviously used as guidance in 1956. Reappraising Oppenheim's early writings delivers two significant payoffs: new clarity in understanding the concept of systematization as well as a more solid grasp of the structure of this approach as a distinctive combination of explanation and systematization. • The concept of systematization in the 1956 KO approach to intertheoretical reduction can be explicated with Oppenheim's philosophy of science from the 1920s and 1930s. • The degree of systematization of a theory is measured by the number of its terms. • There is a direct connection between systematization and explanation. • To explain means to describe something with fewer terms, i.e. to merge a summative juxtaposition of statements into one systematic law. [ABSTRACT FROM AUTHOR]

Solution structure of a heterodimeric CD3epsilongamma ectodomain complex, an integral component of the T cell receptor (TCR) and pre-TCR, reveals that TCR-based signal transduction is facilitated by an hydrophobic interphase between the two C2-set immunoglobulin-like domains and parallel pairing of the C-terminal beta-strands of the complex.

Results describe the structure of CIDE-N of human CIDE-B protein and show its interaction with CIDE-N domains of both 40 kDA capsule-activated DNA fragmentation factor (DFF40) and the inhibitor DFF45. Role of this novel bipolar interaction in the regulation of downstream events of apoptosis is discussed.

A study was conducted to characterize the three-dimensional caspase recruitment domain structure of RAIDD adaptor proteins using nuclear magnetic resonance (NMR) spectroscopy. Varian spectrometers were used to determine NMR spectra while structure computations were carried out based on simulated annealing protocols. Results indicated that the adaptor proteins support a six-helix bundle and distinct charged surface patches.

Protein kinase such as the casein kinase Ialpha and MEKK1 have synergic roles in suppressing NF-AT. MEKK1 protein kinase indirectly suppresses NF-AT4 nuclear import while casein kinase Ialpha directly binds and phosphorylates the transcription factor NF-AT4 which resulted to the inhibition of the transcription factor's nuclear translocation. These transcriptional factors have important function in maintaining and enhancing the masking activity responsible in controlling NF-AT4.

Research was conducted to study the solution composition of the binary complex established between the minimal DNA recognition domain of the human nuclear factor of the activated T cell (NFATC) and an oligonucleotide duplex characterized by the interleukin-2 enhancer sequence. Site-directed mutation was utilized to determine an Escherichia coli overexpression system for NFATC. Results indicated that the gain of DNA-binding affinity can be determined on the basis of the nuclear magnetic resonance complex structure.

Results show that mRNA, the eukaryotic initiation factor 4G (elF4G), and elF4E exist as a stable mRNP, which mediates ribosomal loading during translation initiation in protein synthesis. The solution structure of the complex between yeast elf4E/cap and elF4G is presented.

Research reveals that the tumor suppressor protein, ING2, is a nuclear phosphoinositide receptor and contains a plant homeodomain (PHD) finger. Data indicate that the PHD fingers bind to phosphoinositides including phosphatidylinositol 5-phosphate with which it interacts to regulate ING2 activation of p53 and p53-dependent apoptotic pathways.

Highlights • cNDs represent a significant advance in the applicability and durability of nanodisc technology. • We highlighted the technical challenges that needed to be overcome to obtain high quality cNDs. • Large cNDs can be used to study the early steps in virus infection. Covalently circularized nanodiscs (cNDs) represent a significant advance in the durability and applicability of nanodisc technology. The new cNDs demonstrate higher size homogeneity and improved stability compared with that of non-circularized forms. Moreover, cNDs can be prepared at various defined sizes up to 80-nm diameter. The large cNDs can house much larger membrane proteins and their complexes than was previously possible with the conventional nanodiscs. In order to experience the full advantages of covalent circularization, high quality circularized scaffold protein and nanodisc samples are needed. Here, we give a concise overview and discuss the technical challenges that needed to be overcome in order to obtain high quality preparations. Furthermore, we review some potential new applications for the cNDs. [ABSTRACT FROM AUTHOR]

Tree leaves and needles are commonly used in biomonitoring as reliable indicators of environmental pollution to monitor a wide range of mainly airborne contaminants such as heavy metals and persistent organic pollutants (POPs). The most severe challenges are the reproducibility and comparability of results. This is the only basis upon which correct assessment can be ensured. Standardizing the collection and preservation of leaf samples can overcome such problems and improve the quality of the results. On this account, efforts are being made to promote standardization on national (Association of German Engineers, VDI) and international (European Committee for Standardization, CEN) level. The objective of this review is to investigate the current state of quality assurance in passive biomonitoring by analyzing 86 peer-reviewed papers on tree leaf-sampling and use. We show that little attention has been given to standardizing how leaves are collected in the field and stored as well as to the documentation of selection criteria and sampling conditions. Important information is often absent, including crucial aspects of sample collection and preservation, for example, the selection of sampling sites, exposure, sampling period, age and condition of target compartments. Potential standards are suggested and discussed as a foundation for the development of quality assurance standards to conceptualize and perform sampling. For some aspects of sampling the current literature yields little information to derive and establish standards. In the first place, this concerns the selection of sample species. Besides of this, it is important to document and report data concerning sampling conditions in a publication. Overall, further research is urgently required to define scientifically valid standards, which will ensure a high level of quality and comparability of the samples collected in the field. [ABSTRACT FROM AUTHOR]

A study was conducted to analyze the crystal structure of the heterophilic adhesion complex between the amino-terminal domains of human CD2 and CD58. Results indicated an asymmetric, orthogonal, face-to-face interaction involving the major beta sheets of the respective immunoglobulin-like domains. Findings also showed that charged amino acid side chains form hydrogen bonds and salt links at the interface during the virtual absence of hydrophobic forces.

The solution structure of the cross-talk agent BID is determined. This proapoptosis member of the BCL2 regulatory family is shown to consist of eight alpha helices arranged in a compact fold homologous to BCL-X(sub L). It is also shown that the hydrophobic cleft of BCL-X(sub L), which binds to the BAK BH3 domain, readily interacts with the BH3 domain-containing helix. Structural data on the BCL-X(sub L)-BAK BH3 peptide complex is then used to model the heterodimerization between BCL-X(sub L) and the full-length BID by superimposing the BH3 domains of BAK and BID.

The voltage-dependent anion channel (VDAC) is an essential protein in the eukaryotic outer mitochondrial membrane, providing the pore for substrate diffusion. Three high-resolution structures of the isoform 1 of VDAC in detergent micelles and bicelles have recently been published, using solution NMR and X-ray crystallography. They resolve longstanding discussions about the membrane topology of VDAC and provide the first eukaryotic β-barrel membrane protein structure. The structure contains a surprising feature that had not been observed in an integral membrane protein before: A parallel β-strand pairing and thus an odd number of strands. The studies also give a structural and functional basis for the voltage gating mechanism of VDAC and its modulation by NADH; however, they do not fully explain these functions yet. With the de novo structure of VDAC-1, as well as those of half a dozen other proteins, the number of integral membrane protein structures solved by solution NMR has doubled in the past two years. Numerous further structural and functional studies on many different membrane proteins show that solution NMR has become an important tool for membrane protein molecular biology. [Copyright &y& Elsevier]

Interactions of proteins with other macromolecules or small molecules play important roles in most biological processes. Often, such interactions are weak and transient, and the complexes do not easily crystallize. NMR spectroscopy has the unique ability to retrieve information about these interactions and is increasingly used. Recent methodological developments have helped characterize weak protein interactions, and have in particular been applied to the study of proteins that are mostly unfolded alone but form well-defined complexes upon interaction. In addition, NMR methods have been applied to the identification and characterization of small chemicals that inhibit protein function, a primary objective of rational drug design. [Copyright &y& Elsevier]

An abstract of the article "NMR Methods for Studying Protein-Protein Interactions Involved in Translation Initiation," by Assen Marintchev, Dominique Frueh, and Gerhard Wagner is presented.

The GTP-bound form of the trimeric eukaryotic translation initiation factor 2 (eIF2) transfers aminoacylated initiator methionyl tRNA onto the 40S ribosome. We have solved with solution NMR the structure of the α subunit of human eIF2 (heIF2α). The protein consists of two domains that are mobile relative to each other. The N-terminal domain has an S1-type oligonucleotide/oligosaccharide binding-fold subdomain and an α-helical subdomain. The C-terminal domain adopts an αβ-fold very similar to the C-terminal domain of elongation factor (eEF) 1Bα, the guanine-nucleotide exchange factor for eEF1A. The structural and functional similarities found between eIF2α/eIF2γ and eEF1Bα/eEF1A suggest a model for the interaction of eIF2α with eIF2γ, and eIF2 with Met-tRNAiMet. It further indicates a previously unrecognized evolutionary lineage of eIF2α/γ from the functionally related elongation factor eEF1Bα/eEF1A complex. [Copyright &y& Elsevier]

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 singlestranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 ptimase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork. [ABSTRACT FROM AUTHOR]

Using ICP-MS and ICP-AES platinum group elements (Pt, Pd, Rh, Ru and Ir) and Ce, La, Nd, Pb and Zr have been determined in street dust, Taraxacum officinale (dandelion), Plantago lanceolata (plantain), Lolium multiflorum (annual ryegrass), Rhytidiadelphus squarrosus (moss) and Vascellum pratense (mushrooms) collected along highways and streets in Germany during 1999. Among the plants Taraxacum officinale (dandelion) reflects most adequately the pollution with the investigated elements matching the results from street dust. A strong positive correlation between all elements determined in the plants is established. Transfer factor for Pt between soil and plants has been determined in an agricultural experiment ranging between 0.004 and 0.008 for two types of soils. [Copyright &y& Elsevier]

Changes in the concentration of cosmogenic nuclides measured in natural archives are related mainly to changes in the cosmic-ray-induced production rate in the atmosphere, but also to the subsequent transport and deposition processes which depend on the geochemical properties of the nuclides. A comparison of 10Be and 36Cl data from ice cores from Greenland ice cores and 14C data from tree rings and other archives provides the following information for the isotope stages 2 and 3:

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αβT lymphocytes through their β chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αβTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRβ-pMHC interaction. The linkage model of preTCRβ-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies. [ABSTRACT FROM AUTHOR]

• Identified single domain antibodies (VHHs) that bind to UBC6e with high affinity. • VHH binds to a short linear epitope near a phosphorylation site. • VHH binding to UBC6e enhances enzymatic activity. • VHH binding inhibits UBC6e phosphorylation in cells. A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitro and interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility. [ABSTRACT FROM AUTHOR]

• NMR yields quantitative data on the dynamics of solution-state protein structures. • NMR's impact is enhanced by continuous innovation in assignment techniques. • Improvements in hardware and experimental design have extended the reach of NMR. • NMR provides access to lowly populated invisible states. • Information provided by NMR is unique and complements other biophysical techniques. The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-resolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Here, we summarize recent technical landmarks in solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic resolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics. [ABSTRACT FROM AUTHOR]

The pre-T cell receptor (pre-TCR) guides early thymocytes through maturation processes within the thymus via interaction with self-ligands displayed on thymic epithelial cells. The pre-TCR is a disulfide-linked heterodimer composed of an invariant pre-TCR α (pTα) subunit and a variable β subunit, the latter of which is incorporated into the matureTCRin subsequent developmental progression. This interaction of pre-TCR with peptide-major histocompatibility complex (pMHC) molecules has recently been shown to drive robust pre-TCR signaling and thymocyte maturation. Although the native sequences of β are properly folded and suitable for NMR studies in isolation, a tendency to self-associate rendered binding studies with physiological ligands difficult to interpret. Consequently, to structurally define this critical interaction, we have re-engineered the extracellular regions of β, designated as β-c1, for prokaryotic production to be used inNMRspectroscopy. Given the large size of the full extracellular domain of class IMHCmolecules such as H-Kb, we produced a truncated form termed Kb-t harboring properties favorable for NMR measurements. This system has enabled robust measurement of a pre-TCR-pMHC interaction directly analogous to that of TCRαβ-pMHC. Binding surface analysis identified a contact surface comparable in size to that of the TCRαβ-pMHC but potentially with a rather distinct binding orientation. A tilting of the pre-TCRβ when bound to the pMHCligand recognition surface versus the upright orientation of TCRαβ would alter the direction of force application between pre-TCR and TCR mechanosensors, impacting signal initiation. [ABSTRACT FROM AUTHOR]

The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre- TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the αβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. [ABSTRACT FROM AUTHOR]

Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10-31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp21 and Trp25) and right (harboring Trp29) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp21 and Trp25 and two adjacent leucines. Trp29 serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm. [ABSTRACT FROM AUTHOR]

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function. [ABSTRACT FROM AUTHOR]

The serine/threonine phosphatase calcineurin (Cn) targets the nuclear factors of activated T cells (NFATs) that activate cytokine genes. Calcium influx activates Cn to dephosphorylate multiple serine residues within the ~200 residue NFAT regulatory domain, which triggers joint nuclear translocation of NFAT and Cn. The dephosphorylation process relies on the interaction between Cn and the conserved motifs PxIxIT and LxVP, which are located N- and C-terminal to the phosphorylation sites in NFAT's regulatory domain. Here, we show that an NFATc1-derived 15-residue peptide segment containing the conserved LxVP motif binds to an epitope on Cn's catalytic domain (CnA), which overlaps with the previously established PxIxIT binding site on CnA and is distant to the regulatory domain (CnB). Both NFAT motifs partially compete for binding but do not fully displace each other on the CnA epitope, revealing that both segments bind simultaneously to the same epitope on the catalytic domain. [ABSTRACT FROM AUTHOR]

The CD3εγ and CD3εδ heterodimers along with the CD3ζζ homodimer are the signaling components of the T cell receptor (TCR). These invariant dimers are non-covalently associated on the T cell plasma membrane with a clone-specific (i.e. clonotypic) αβ heterodimer that binds its cognate ligand, a complex between a particular antigenic peptide, and an MHC molecule (pMHC). These four TCR dimers exist in a 1:1:1:1 stoichiometry. At the junction between the extracellular and transmembrane domains of each mammalian CD3ε, CD3γ, and CD3δ subunit is a highly conserved CXXC motif previously found to be important for thymocyte and T cell activation. The redox state of each CXXC motif is presently unknown. Here we show using LC-MS and a biotin switch assay that these CXXC segments are constitutively oxidized on resting and activated T cells, consistent with their measured reduction potential. NMR chemical shift perturbation experiments comparing a native oxidized CD3δ CXXC-containing segment with that of a mutant SXXS-containing CD3δ segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1'-racglycerol) (sodium salt)) micelles show extensive chemical shift differences in residues within the membrane-proximal motif as well as throughout the transmembrane and cytoplasmic domains as a result of the elimination of the native disulfide. Likewise, direct comparison of the native CD3δ segment in oxidizing and reducing conditions reveals numerous spectral differences. The oxidized CXXC maintains the structure within the membrane-proximal stalk region as well as that of its contiguous transmembrane and cytoplasmic domain, inclusive of the ITAM (immunoreceptor tyrosine-based activation motif) involved in signaling. These results suggest that preservation of the CD3 CXXC oxidized state may be essential for TCR mechanotransduction. [ABSTRACT FROM AUTHOR]

Abstract: Protein–protein interactions are critical for regulating the activity of translation initiation factors and multitude of other cellular process, and form the largest block of untapped albeit most challenging targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule libraries as an inhibitor of translation initiation factors eIF4E and eIF4G protein–protein interaction; it inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have been designed and synthesized in order to delineate the structural latitude and improve its binding affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding affinity and translation initiation inhibitory activities. [Copyright &y& Elsevier]

Background: Despite analyses of broadly neutralizing anti-HIV-1 antibodies directed against the gp41 MPER segment, there exists a paucity of structural information on MPER immunogenicity. Results: Immunodominance of Trp-680 in the MPER arrayed on liposomes is modified by membrane anchoring. Conclusion: Immunogenicity is manipulatable through subtle structural modification. Significance: Learning about the structural basis of immunogenicity is critical for eliciting desired B cell antibody production through vaccination. [ABSTRACT FROM AUTHOR]

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui- phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui- mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd- phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex. [ABSTRACT FROM AUTHOR]

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF- 1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels. [ABSTRACT FROM AUTHOR]

Summary: To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing microtubules, which undergo dramatic structural changes forming curved protofilaments, has yet to be defined in vertebrates. Here, we demonstrate that the conserved kinetochore-localized Ska1 complex tracks with depolymerizing microtubule ends and associates with both the microtubule lattice and curved protofilaments. In contrast, the Ndc80 complex, a central player in the kinetochore-microtubule interface, binds only to the straight microtubule lattice and lacks tracking activity. We demonstrate that the Ska1 complex imparts its tracking capability to the Ndc80 complex. Finally, we present a structure of the Ska1 microtubule-binding domain that reveals its interaction with microtubules and its regulation by Aurora B. This work defines an integrated kinetochore-microtubule interface formed by the Ska1 and Ndc80 complexes that associates with depolymerizing microtubules, potentially by interacting with curved microtubule protofilaments. [ABSTRACT FROM AUTHOR]

Abstract: Three isoforms of the human voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, are crucial regulators of mitochondrial function. Numerous studies have been carried out to elucidate biochemical properties, as well as the three-dimensional structure of VDAC-1. However, functional and structural studies of VDAC-2 and VDAC-3 at atomic resolution are still scarce. VDAC-2 is highly similar to VDAC-1 in amino acid sequence, but has substantially different biochemical functions and expression profiles. Here, we report the reconstitution of functional VDAC-2 in lauryldimethylamine-oxide (LDAO) detergent micelles and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer nanodiscs. We find that VDAC-2 is properly folded in both membrane-mimicking systems and that structural and functional characterization by solution NMR spectroscopy is feasible. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism. [Copyright &y& Elsevier]

DISC1 (Disrupted in schizophrenia-1) plays essential roles in neuronal proliferation, neuronal migration and axon guidance and has been implicated in schizophrenia and related psychiatric disorders. DISC1 forms a functional complex with nuclear distribution element-like protein-1 (NDEL1), a key component that regulates microtubule organization during cell division and neuronal migration. DISC1 polymorphisms at the binding interface of DISC1-NDEL1 complex have been implicated in schizophrenia. However, it is unknown how schizophrenia risk polymorphisms perturb its interaction with NDEL1 and how they change the inherent biochemical properties of DISC1. Here, we characterize the oligomerization and binding property of DISC1 and its natural schizophrenia risk variant, S704C. Our results show that DISC1 forms octamers via dimers as building blocks and directly interacts with tetramers of NDEL1. The schizophrenia risk variant S704C affects the formation of octamers of DISC1 and exhibits higher-order self-oligomerization. However, the observed formation of new oligomeric species did not influence its binding with NDEL1. These results suggest that the improper oligomeric assembly of DISC1-5704C may underlie the observed phenotypic variation due to the polymorphism. [ABSTRACT FROM AUTHOR]

The most abundant protein of the mitochondrial outer membrane is the voltage-dependent anion channel (VDAC), which facilitates the exchange of ions and molecules between mitochondria and cytosol and is regulated by interactions with other proteins and small molecules. VDAC has been studied extensively for more than three decades, and last year three independent investigations revealed a structure of VDAC-1 exhibiting 19 transmembrane β-strands, constituting a unique structural class of β-barrel membrane proteins. Here, we provide a historical perspective on VDAC research and give an overview of the experimental design used to obtain these structures. Furthermore, we validate the protein refolding approach and summarize the biochemical and biophysical evidence that links the 19-stranded structure to the native form of VDAC. [Copyright &y& Elsevier]

Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, nonmicellar solubilization techniques, with a particular focus on their application to solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes. [ABSTRACT FROM AUTHOR]

Thymus-derived lymphocytes protect mammalian hosts against virusor cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric αβ, CD3ϵγ, CD3ϵδ, and CD3ζζ subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3ϵ lobe that they approach diagonally, adjacent to the lever-like Cβ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3ϵ and CD3γ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (∼50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands. [ABSTRACT FROM AUTHOR]