Chmp4c is required for stable kinetochore-microtubule attachments

Formation of stable kinetochore-microtubule attachments is essential for accurate chromosome segregation in human cells and depends on the NDC80 complex. We recently showed that Chmp4c, an endosomal sorting complex required for transport protein involved in membrane remodelling, localises to prometaphase kinetochores and promotes cold-stable kinetochore microtubules, faithful chromosome alignment and segregation. In the present study, we show that Chmp4c associates with the NDC80 components Hec1 and Nuf2 and is required for optimal NDC80 stability and Hec1-Nuf2 localisation to kinetochores in prometaphase. However, Chmp4c-depletion does not cause a gross disassembly of outer or inner kinetochore complexes. Conversely, Nuf2 is required for Chmp4c kinetochore targeting. Constitutive Chmp4c kinetochore tethering partially rescues cold-stable microtubule polymers in cells depleted of the endogenous Nuf2, showing that Chmp4c also contributes to kinetochore-microtubule stability independently of regulating Hec1 and Nuf2 localisation. Chmp4c interacts with tubulin in cell extracts, and binds and bundles microtubules in vitro through its highly basic N-terminal region (amino acids 1–77). Furthermore, the N-terminal region of Chmp4c is required for cold-stable kinetochore microtubules and efficient chromosome alignment. We propose that Chmp4c promotes stable kinetochore-microtubule attachments by regulating Hec1–Nuf2 localisation to kinetochores in prometaphase and by binding to spindle microtubules. These results identify Chmp4c as a novel protein that regulates kinetochore-microtubule interactions to promote accurate chromosome segregation in human cells.     

The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis

Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the "tail." Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore-microtubule attachment in distinct ways. The most severe defects in kinetochore-microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore-microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.     

Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable "core" region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.     

Structure of a central component of the yeast kinetochore: the Spc24p/Spc25p globular domain

The Ndc80 complex, a kinetochore component conserved from yeast to humans, is essential for proper chromosome alignment and segregation during mitosis. It is an approximately 570 A long, rod-shaped assembly of four proteins--Ndc80p (Hec1), Nuf2p, Spc24p, and Spc25p--with globular regions at either end of a central shaft. The complex bridges from the centromere-proximal inner kinetochore layer at its Spc24/Spc25 globular end to the microtubule binding outer kinetochore layer at its Ndc80/Nuf2 globular end. We report the atomic structures of the Spc24/Spc25 globular domain, determined both by X-ray crystallography at 1.9 A resolution and by NMR. Spc24 and Spc25 fold tightly together into a single globular entity with pseudo-2-fold symmetry. Conserved residues line a common hydrophobic core and the bottom of a cleft, indicating that the functional orthologs from other eukaryotes will have the same structure and suggesting a docking site for components of the inner kinetochore.     

Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus.

Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C. We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs. Eight residues were identified as important for triphosphatase activity. These were Glu-305, Glu-307, and Phe-310 in motif A (IELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-496 in motif C (EVELE). Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p. E492Q retained triphosphatase activity. Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate. Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K(m) for ATP, whereas substitution with lysine increased K(m) 6-fold. Changing Lys-456 to alanine or glutamine increased K(m) an order of magnitude; ATP binding was restored when arginine was introduced. Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function. Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu-495 resulted in thermal instability in vivo and in vitro. A second S. cerevisiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized. Cth1p activity was abolished by E87A and E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo. Yet, fusion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1Delta yeast cells. This finding illustrates that mammalian guanylyltransferase can be used as a vehicle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through its binding to the phosphorylated CTD of RNA polymerase II.     

The vertebrate Ndc80 complex contains Spc24 and Spc25 homologs, which are required to establish and maintain kinetochore-microtubule attachment

How kinetochores bind to microtubules and move on the mitotic spindle remain unanswered questions. Multiple systems have implicated the Ndc80/Hec1 (Ndc80) kinetochore complex in kinetochore-microtubule interaction and spindle checkpoint activity. In budding yeast, Ndc80 copurifies with three additional interacting proteins: Nuf2, Spc24, and Spc25. Although functional vertebrate homologs of Ndc80 and Nuf2 exist, extensive sequence similarity searches have not uncovered homologs of Spc24 and Spc25. We have purified the xNdc80 complex to homogeneity from Xenopus egg extracts and identified two novel interacting proteins. Although the sequences have greatly diverged, we have concluded that these are the functional homologs of the yeast Spc24 and Spc25 proteins based on limited sequence similarity, common coiled-coil domains, kinetochore localization, similar phenotypes, and copurification with xNdc80 and xNuf2. Using both RNAi and antibody injection experiments, we have extended previous characterization of the complex and found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetochore-microtubule attachments and metaphase alignment. In addition, we show that Spc24 and Spc25 are required for chromosomal movement to the spindle poles in anaphase.     

Membrane topography of ColE1 gene products: the immunity protein.

The topography of the colicin E1 immunity (Imm) protein was determined from the positions of TnphoA and complementary lacZ fusions relative to the three long hydrophobic segments of the protein and site-directed substitution of charged for nonpolar residues in the proposed membrane-spanning segments. Inactivation of the Imm protein function required substitution and insertion of two such charges. It was concluded that the 113-residue colicin E1 Imm protein folds in the membrane as three trans-membrane alpha-helices, with the NH2 and COOH termini on the cytoplasmic and periplasmic sides of the membrane, respectively. The approximate spans of the three helices are Asn-9 to Ser-28, Ile-43 to Phe-62, and Leu-84 to Leu-104. An extrinsic highly charged segment, Lys-66 to Lys-74, containing seven charges in nine residues, extends into the cytoplasmic domain. The specificity of the colicin E1 Imm protein for interaction with the translocation apparatus and the colicin E1 ion channel is proposed to reside in its peripheral segments exposed on the surface of the inner membrane. These regions include the highly charged segment Lys-66 to Lys-83 (loop 2) and the short (approximately eight-residue) NH2 terminus on the cytoplasmic side, and Glu-29 to Val-44 (loop 1) and the COOH-terminal segment Gly-105 to Asn-113 on the periplasmic side.     

Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity

Microtubule (MT) attachment to kinetochores is vitally important for cell division, but how these interactions are controlled by phosphorylation is not well known. We used quantitative approaches in vitro combined with molecular dynamics simulations to examine phosphoregulation of the NDC80 complex, a core kinetochore component. We show that the outputs from multiple phosphorylation events on the unstructured tail of its Hec1 subunit are additively integrated to elicit gradual tuning of NDC80-MT binding both in vitro and in silico. Conformational plasticity of the Hec1 tail enables it to serve as a phosphorylation-controlled rheostat, providing a new paradigm for regulating the affinity of MT binders. We also show that cooperativity of NDC80 interactions is weak and is unaffected by NDC80 phosphorylation. This in vitro finding strongly supports our model that independent molecular binding events to MTs by individual NDC80 complexes, rather than their structured oligomers, regulate the dynamics and stability of kinetochore-MT attachments in dividing cells.     

Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.     

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.     

Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nM, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine beta-trypsin were obtained that diffracted to 1.8 A. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Ogamma of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1.     

The functional region of CENP-H interacts with the Nuf2 complex that localizes to centromere during mitosis

CENP-H is a constitutive centromere component that localizes to the centromere throughout the cell cycle. Because CENP-H is colocalized with CENP-A and CENP-C, it is thought to be an inner centromere protein. We previously generated a conditional loss-of-function mutant of CENP-H and showed that CENP-H is required for targeting of CENP-C to the centromere in chicken DT40 cells. In the present study, we used this mutant to identify the functional region of chicken CENP-H necessary for centromere targeting and cell viability. This region was found by yeast two-hybrid analysis to interact with Hec1, which is a member of the Nuf2 complex that transiently localizes to the centromere during mitosis. Coimmunoprecipitation experiments revealed that CENP-H interacts with the Nuf2 complex in chicken DT40 cells. Photobleaching experiments showed that both Hec1 and CENP-H form stable associations with the centromeres during mitosis, suggesting that Hec1 acts as a structural component of centromeres during mitosis. On the basis of these results and previously published data, we propose that the Nuf2 complex functions as a connector between the inner and outer kinetochores.     

The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation

We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.     

Synthesis and biological evaluation of N-aryl-2-phenyl-hydrazinecarbothioamides: Experimental and theoretical analysis on tyrosinase inhibition and interaction with HSA

A series of N-aryl-2-phenyl-hydrazinecarbothioamides have been investigated as possible inhibitors of tyrosinase, an enzyme involved in the development of melanomas. The hydrazinecarbothioamides 1–6 were synthesized from the reaction between phenylhydrazine and isothiocyanates, for which three different methods have been employed, namely stirring at room temperature, by microwave irradiation or by mechanochemical grinding. Quantitative yields were obtained for the later technique. Compound 4 showed the best value for tyrosinase inhibition (IC₅₀ = 22.6 µM), which occurs through an uncompetitive mechanism. Molecular docking results suggested that 4 can interact via T-stacking with the substrate L-DOPA and via hydrogen bonding and hydrophobic forces with the amino acid residues Ala-79, His-243, Val-247, Phe-263, Val-282, and Glu-321. The interaction between human serum albumin (HSA) and compound 4 occurs through a ground state association and does not perturb the secondary structure of the albumin as well as the microenvironment around Tyr and Trp residues. The binding is spontaneous, moderate and occurs mainly in the Sudlow’s site I. Molecular docking results suggested hydrogen bonding, hydrophobic and electrostatic interactions as the main binding forces between the compound 4 and the amino acid residues Lys-198, Trp-214, Glu-449, Leu-452, and Leu-480.     

Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore

The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex.     

Induced fit of an epitope peptide to a monoclonal antibody probed with a novel parallel surface plasmon resonance assay

Class II major histocompatibility complex proteins bind peptides for presentation to T-cells as part of the immune response process. Monoclonal antibody MEM-265 recognizes the peptide-free conformation of the major histocompatibility complex class II protein HLA-DR1 through specific binding to an epitope contained between residues 50-67 of the beta-chain. In previous work using alanine scanning (1), we identified residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 as essential for specific recognition by MEM-265. The spacing of these residues approximates a 3.5-residue repeat, suggesting that MEM-265 may recognize the epitope in an alpha-helical conformation. In the folded, peptide-loaded DR1 structure, the beta-chain residues 50-67 contain a kinked alpha-helical segment spanning Glu-52-Ser-63 (2). However, the conformation of this segment in the peptide-free form is unknown. We have used a new surface plasmon resonance approach in a SpotMatrix format to compare the kinetic rates and affinities for 18 alanine scanning mutants comprising epitope residues 50-67. In addition to the six essential residues described previously, we found two additional residues, Glu-52 and Gln-64, that contribute by enhancing MEM-265 binding. By contrast, mutation of either Gly-54 or Pro-56 to an alanine actually improved binding to MEM-265. In essentially all cases peptide substitutions that either improve or reduce MEM-265 recognition could be traced to differences in the dissociation rate (k off). The kinetic details of the present study support the presence of a structural component in the antigenic epitope recognized by MEM-265 in the peptide-free form of major histocompatibility complex II DR1 beta-chain.     

Role of conserved surface amino acids in binding of SmpB protein to SsrA RNA

Bacteria possess a unique salvage mechanism for rescuing ribosomes stalled on aberrant mRNAs. A complex of SmpB protein and SsrA RNA orchestrates this salvage process. The specific and direct binding of SmpB facilitates recognition and delivery of SsrA RNA to stalled ribosomes. The SmpB protein is conserved throughout the bacterial kingdom and contains several conserved amino acid sequence motifs. We present evidence to demonstrate that amino acid residues Glu-31, Leu-91, and Lys-124, which are highly conserved and clustered along an exposed surface of the protein, play a crucial role in the SsrA-mediated peptide tagging process. Our analysis suggests that the peptide-tagging defect exhibited by these SmpB variants is due to their inability to facilitate the delivery of SsrA RNA to stalled ribosomes. Moreover, we present evidence to demonstrate that the ribosome association defect of these variants is due to their reduced SsrA binding affinity. Consistent with these findings, we present biochemical evidence to demonstrate that residues Glu-31, Leu-91, and Lys-124 are essential for the SsrA binding activity of SmpB protein. Furthermore, we have investigated the interactions of SmpB.SsrA orthologues from the thermophilic bacterium Thermoanaerobacter tengcongensis. Our investigations demonstrate an analogous role for the equivalent T. tengcongensis residues in SmpB.SsrA interactions, hence further validating our findings for the Escherichia coli SmpB.SsrA system. These results demonstrate the functional significance of this cluster of conserved residues in SmpB binding to SsrA RNA, suggesting they might represent a core contact surface for recognition of SsrA RNA.     

Identification of amino acid residues critical for biological activity in human interleukin-18

Interleukin-18 (IL-18) is a pro-inflammatory cytokine, and IL-18-binding protein (IL-18BP) is a naturally occurring protein that binds IL-18 and neutralizes its biological activities. Computer modeling of human IL-18 identified two charged residues, Glu-42 and Lys-89, which interact with oppositely charged amino acid residues buried in a large hydrophobic pocket of IL-18BP. The cell surface IL-18 receptor alpha chain competes with IL-18BP for IL-18 binding, although the IL-18 receptor alpha chain does not share significant homology to IL-18BP. In the present study, Glu-42 was mutated to Lys and Lys-89 to Glu; Glu-42 and Lys-89 were also deleted separately. The deletion mutants (E42X and K89X) were devoid of biological activity, and the K89E mutant lost 95% of its activity. In contrast, compared with wild-type (WT) IL-18, the E42K mutant exhibited a 2-fold increase in biological activity and required a 4-fold greater concentration of IL-18BP for neutralization. The binding of WT IL-18 and its various mutants to human natural killer cells was evaluated by competition assays. The mutant E42K was more effective than WT IL-18 in inhibiting the binding of (125)I-IL-18 to natural killer cells, whereas the three inactive mutants E42X, K89E, and K89X were unable to compete with (125)I-IL-18 for binding. Similarly, WT IL-18 and the E42K mutant induced degradation of Ikappa-Balpha, whereas the three biologically inactive mutants did not induce degradation. The present study reveals that Glu-42 and Lys-89 are critical amino acid residues for the integrity of IL-18 structure and are important for binding to cell surface receptors, for signal transduction, and for neutralization by IL-18BP.     

The human kinetochore proteins Nnf1R and Mcm21R are required for accurate chromosome segregation

Kinetochores (KTs) assemble on centromeric DNA, bi-orient paired sister chromatids on spindle microtubules (MTs) and control cell-cycle progression via the spindle assembly checkpoint. Genetic and biochemical studies in budding yeast have established that three 'linker' complexes, MIND, COMA and NDC80, play essential but distinct roles in KT assembly and chromosome segregation. To determine whether similar linker activities are present at human KTs, we have compared the functions of Nnf1R and Mcm21R, recently identified MIND and COMA subunits, and Nuf2R, a well-characterized NDC80 subunit. We find that the three proteins bind to KTs independent of each other and with distinct cell-cycle profiles. MT-KT attachment is aberrant in Nnf1R- and Mcm21R-depleted cells, whereas it is lost in the absence of Nuf2R. Defective attachments in Nnf1R-depleted cells prevent chromosome congression, whereas those in Mcm21R-depleted cells interfere with spindle assembly. All three human KT proteins are necessary for correct binding of spindle checkpoint proteins to KTs. The differing functions and KT-binding properties of Nnf1R, Mcm21R and Nuf2R suggest that, like their yeast counterparts, the proteins act independent of each other in KT assembly, but that their combined activities are required for checkpoint signaling.     

Inhibition of xanthine oxidase by theaflavin: Possible mechanism for anti-hyperuricaemia effect in mice

Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Excessive production of uric acid leads to hyperuricaemia. Due to the serious side effects of allopurinol, it is an urgent need to explore new XO inhibitors. Herein, the effects of theaflavin (TF1) on XO and anti-hyperuricaemia effect in hyperuricemic mice were investigated. Kinetic analysis indicate that TF1 is a reversible competitive inhibitor and has a significant inhibitory effect on XO with an IC₅₀ value of 63.17 ± 0.13 μmol/L. Analysis of fluorescence spectra suggests that TF1 causes the obvious fluorescence quenching of XO, which is mainly driven by hydrophobic interactions and hydrogen bonds. Docking studies demonstrate that TF1 interacts with dozens of amino acid residues surrounded in the active cavity of XO, including Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802, Pro-1076, Leu-873, Leu-1014, Asn-768, Leu-648 and Phe-649. The inhibitory mechanism may be the insertion of TF1 into the active site of XO, which hinders the substrate xanthine to enter into the site. Furthermore, the results from animal experiments demonstrate that TF1 is effective in reducing serum uric acid in mice. These findings suggest that TF1 may be a potential drug candidate for the treatment of hyperuricaemia.