Biomedical subjects

Huilin Li

Publications and source records attributed to Huilin Li.

At least 19 recordsLinked to original sources

PubMed2026

Mechanistic diversity of clamp loading at small DNA gaps.

DNA sliding clamps, including PCNA (proliferating cell nuclear antigen) and the 9-1-1 (RAD9-RAD1-HUS1 in humans) complex, are ring-shaped protein complexes that encircle DNA and serve as central interaction platforms in DNA replication, repair, and checkpoint signaling. While clamp loading at canonical primer-template junctions by AAA+ (ATPases associated with diverse cellular activities) clamp loaders is well established, how clamps are loaded onto physiologically relevant but geometrically constrained DNA intermediates, such as nicks and single-stranded gaps, has remained unclear. Recent cryo-electron microscopy studies reveal that clamp loaders have evolved distinct strategies to overcome these constraints and to specialize for different genomic contexts. At gapped DNA, the eukaryotic clamp loader RFC (replication factor C) engages both 3'- and 5'-recessed DNA ends and can locally unwind DNA, enabling PCNA loading across a wide range of gap sizes. In contrast, the bacterial DnaX clamp loader lacks a 5'-DNA-binding site and does not unwind DNA, instead loading the β-clamp at small gaps (<6 nt) by sharply bending DNA. The checkpoint clamp loader Rad24-RFC (RAD17-RFC in humans) similarly lacks DNA unwinding activity, restricting loading of 9-1-1 clamp to larger gaps (≥6 nt). In a distinct specialization, Ctf18-RFC interacts with the leading-strand DNA polymerase ε, positioning it as a dedicated loader for leading-strand synthesis, whereas Elg1-RFC (ATAD5-RFC in humans) excludes DNA from its chamber and functions as a PCNA unloader. Together, these mechanisms illustrate how clamp loaders are diversified to accommodate DNA structure and replisome context, ensuring coordinated control of genome replication and maintenance.

9-1-1 clamp↗

PubMed2006

Deprotonations and charges of well-defined {Mo72Fe30} nanoacids simply stepwise tuned by pH allow control/variation of related self-assembly processes.

The solution behavior of the largest inorganic acid known thus far, the neutral, spherical iron/molybdenum/oxide nanocluster {Mo72Fe30} ([triple bond{(MoVI) MoVI5}12FeIII30 1a), including the pH-controlled deprotonation, is reported. The acidic properties are due to the 30 peripheral, weakly acidic FeIII(H2O) groups that form a unique Archimedean solid with all edges and dihedral angles being equal, the icosidodecahedron, and therefore an "isotropic" surface. Interestingly, the aqueous solutions are stable even for months because of the inertness of the spherical solutes and the presence of the hard FeIII and MoVI centers. The stability can be nicely proven by the very characteristic Raman spectrum showing, because of the (approximately) icosahedral symmetry, only a few lines. Whereas the {Mo72Fe30} clusters exist as discrete, almost neutral, molecules in aqueous solution at pH < 2.9, they get deprotonated and self-associate into single-layer blackberry-type structures at higher pH while the assembly process (i.e., the size of the final species) can be controlled by the pH values; this allows the deliberate generation of differently sized nanoparticles, a long-term goal in nanoscience. The average hydrodynamic radius (Rh) of the self-assembled structures decreases monotonically with increasing number of charges on the {Mo72Fe30} macroanions (from approximately 45 nm at pH approximately 3.0 to approximately 15 nm at pH approximately 6.6), as studied by laser light scattering and TEM techniques. The {Mo72Fe30} macroions with high-stability tunable charges/surfaces, equal shape, and masses provide models for the understanding of more complex polyelectrolyte solutions while the controllable association and dissociation reported here of the assembled soft magnetic materials with tuneable sizes could be interesting for practical applications.

Journal Article↗

PubMed2006

Dimeric organization of the yeast oligosaccharyl transferase complex.

The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of approximately 500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-A resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation.

Animals↗

PubMed2006

Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42.

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.

Amino Acid Sequence↗

PubMed2006

Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores.

Gamma-secretase is an intramembrane-cleaving aspartyl protease required for the normal development of metazoans because it processes Notch within cellular membranes to release its signaling domain. More than two dozen additional substrates of diverse functions have been reported, including the Notch ligands Delta and Jagged, N- and E-cadherins, and a sodium channel subunit. The protease is causally implicated in Alzheimer's disease because it releases the neurotoxic amyloid beta-peptide (Abeta) from its precursor, APP. Gamma-secretase occurs as a large complex containing presenilin (bearing the active site aspartates), nicastrin, Aph-1, and Pen-2. Because the complex contains at least 18 transmembrane domains, crystallographic approaches to its structure are difficult and remote. We recently purified the human complex essentially to homogeneity from stably expressing mammalian cells. Here, we use EM and single-particle image analysis on the purified enzyme, which produces physiological ratios of Abeta40 and Abeta42, to obtain structural information on an intramembrane protease. The 3D EM structure revealed a large, cylindrical interior chamber of approximately 20-40 A in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer. Lectin tagging of the nicastrin ectodomain enabled proper orientation of the globular, approximately 120-A-long complex within the membrane and revealed approximately 20-A pores at the top and bottom that provide potential exit ports for cleavage products to the extra- and intracellular compartments. Our reconstructed 3D map provides a physical basis for hydrolysis of transmembrane substrates within a lipid bilayer and release of the products into distinct subcellular compartments.

Amyloid Precursor Protein Secretases↗

PubMed2006

Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity.

Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's beta-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg(2+) or Ca(2+). Electron microscopy revealed what appeared to be obstructed alpha-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's alpha-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.

Bacterial Proteins↗

PubMed2006

Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate.

Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 A resolution reveals a substrate-binding pocket with composite features of the distinct beta1, beta2 and beta5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the alpha-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapetides of the alpha-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-beta-(1-naphthyl)-L-alanine-L-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

Binding Sites↗

PubMed2005

Structure of the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans.

Interplay among four genes--egl-1, ced-9, ced-4 and ced-3--controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4-CED-9 complex at 2.6 A resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.

Adenosine Triphosphate↗

PubMed2005

Structural role of glycine in amyloid fibrils formed from transmembrane alpha-helices.

Amyloid fibrils associated with diseases such as Alzheimer's are often derived from the transmembrane helices of membrane proteins. It is known that the fibrils have a cross-beta-sheet structure where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. However, the structural basis for how the membrane-spanning helix is converted into a beta-sheet or how protofibrils associate into fibrils is not known. Here, we use a model peptide corresponding to a portion of the single transmembrane helix of glycophorin A to investigate the structural role of glycine in amyloid-like fibrils formed from transmembrane helices. Glycophorin A contains a GxxxG motif that is found in many transmembrane sequences including that of the amyloid precursor protein and prion protein. We propose that glycine, which mediates helix interactions in membrane proteins, also provides key packing motifs when it occurs in beta-sheets. We show that glycines in the glycophorin A transmembrane helix promote extended beta-strand formation when the helix partitions into aqueous environments and stabilize the packing of beta-sheets in the formation of amyloid-like fibrils. We demonstrate that fibrillization can be disrupted with a new class of inhibitors that target the molecular grooves created by glycine.

Amino Acid Sequence↗

PubMed2005

Mechanochemical degradation kinetics of high-density polyethylene melt and its mechanism in the presence of ultrasonic irradiation.

In this paper, the effect of ultrasonic intensity on the degradation of high-density polyethylene (HDPE) melt, degradation mechanism, ultrasonic degradation kinetics of HDPE melt as well as the development of molecular weight distribution of HDPE melt during ultrasonic degradation were studied. In the initial stage, the ultrasonic degradation of HDPE melt shows a random scission process, and the molecular weight distribution broadens. After that, the ultrasonic degradation of HDPE melt shows a nonrandom scission process, and the molecular weight distribution of HDPE melt narrows with ultrasonic irradiation time. The average molecular weight of HDPE decreases with the increase of ultrasonic intensity and increases and trends forward that of undegraded HDPE with the increase of distance from ultrasonic probe tip, indicating that attenuation of ultrasonic intensity in HDPE melt is very quick. Ultrasonic degradation kinetics of HDPE melt obeys the equation: Mt=M(infinity) + Ae(-kt). The theoretic calculation by this equation accords well with the experimental results. The plausible ultrasonic degradation mechanism of polymer melt based on molecular relaxation was also proposed in this paper.

Journal Article↗

PubMed2005

KChIP2 modulates the cell surface expression of Kv 1.5-encoded K(+) channels.

The Kv channel interacting proteins (KChIPs) were identified in a yeast two hybrid screen using the N terminus of Kv4.3 as bait. Previous studies have demonstrated that KChIP2 associates with voltage-gated K(+) (Kv) pore-forming (alpha) subunits of the Kv4 subfamily and contributes to the formation of the rapidly inactivating and recovering Kv4-encoded cardiac transient outward K(+) channels, I(to,f). Here, we report that co-expression of KChIP2 (or KChIP1) also modulates the functional cell surface expression of Kv1.5-encoded K(+) channels in transiently transfected HEK-293 cells. In contrast to the effects of KChIP2 on Kv4 channels, however, co-expression of KChIP2 (or KChIP1) decreases Kv1.5-encoded K(+) currents. Although current densities are reduced, KChIP2 (or KChIP1) co-expression does not affect the time- or voltage-dependent properties of heterologously expressed Kv1.5-encoded K(+) currents. Immunohistochemical and cell surface biotinylation experiments demonstrate that KChIP2 reduces the cell surface expression of Kv1.5, likely by inhibiting forward trafficking from the endoplasmic reticulum. In addition, biochemical experiments reveal that KChIP2 co-immunoprecipitates with Kv1.5 (as well as Kv4.2/Kv4.3) from adult mouse ventricles, demonstrating that, similar to other Kv accessory subunits, KChIP2 is a multifunctional Kv channel accessory subunit. Taken together, the results here suggest that KChIP2 contributes to the formation of functional mouse ventricular (Kv1.5-encoded) I(K,slow1) channels as well, perhaps, as other Kv1.5-encoded K(+) currents, including I(Kur) (I(K,ultrarapid)), in human atria.

Animals↗

PubMed2005

ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA.

Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC-Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.

Amino Acid Sequence↗

PubMed2005

Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue.

A screen for Mycobacterium tuberculosis (Mtb) mutants sensitive to reactive nitrogen intermediates identified transposon insertions in the presumptive proteasomal ATPase gene mpa (mycobacterium proteasome ATPase; Rv2115c). mpa mutants are attenuated in both wild type and nitric oxide synthase 2 deficient mice. In this work, we show that attenuation of mpa mutants is severe, and that Mpa is an ATPase associated with various cellular activities (AAA) ATPase that forms hexameric rings resembling the eukaryotic complex p97/valosin-containing protein (VCP). Point mutations in the conserved Walker box ATPase motifs of Mpa greatly reduced or abolished ATPase activity in vitro and abrogated protection of Mtb against acidified nitrite. A mutant Mpa protein missing only its last two amino acids retained ATPase activity, yet failed to protect Mtb against nitrite. The corresponding strain was attenuated in mice. Thus, Mpa is an ATPase whose enzymatic activity is necessary but not sufficient to protect against reactive nitrogen intermediates.

Adenosine Triphosphatases↗

PubMed2005

[Study on characteristics of bias caused by flow injection-capillary electrophoresis with split-flow electrokinetic injection].

The characteristics of bias caused by split-flow electrokinetic injection (SEKI), a new sample injection method applied in coupled flow injection-capillary electrophoretic system (FI-CE), was investigated using pseudoephedrine hydrochloride, a basic drug and ibuprofen, an acidic drug, as model analytes. It was found that bias imposed by SEKI under the conditions of continuous sample matrix/running buffer was similar to that did by electrokinetic injection (EKI). The linearity of calibration curve provided by SEKI was similar to that offered by non-bias hydrodynamic injection (HDI) but significantly better than that obtained by EKI. These features were exploited to improve analytical performances in simultaneous determination of the minor ingredient of pseudoephedrine hydrochloride and the major ingredient of ibuprofen in pharmaceutical preparation. Detectability of 0.7 mg/L for pseudoephedrine hydrochloride, was achieved at a sample throughput rate of 24 times per hour which is 30% lower than that obtained by HDI-based conventional CE. Eleven runs of a test solution containing 13.1 mg/L pseudoephedrine hydrochloride and 81.4 mg/L ibuprofen produced relative standard deviations (RSDs) of 2.8% for the minor ingredient and 1.2% for the major ingredient, which were improved in comparision to that obtained by HDI-based conventional CE. Analytical results for two batches of compound ibuprofen tablets by the SEKI-based FI-CE approach were in good agreement with that obtained by a conventional high performance liquid chromatographic method.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2004

The outer membrane usher forms a twin-pore secretion complex.

The PapC usher is an outer membrane protein required for assembly and secretion of P pili in uropathogenic Escherichia coli. P pilus biogenesis occurs by the chaperone/usher pathway, a terminal branch of the general secretory pathway. Periplasmic chaperone-subunit complexes target to the PapC usher for fiber assembly and secretion through the usher to the cell surface. The molecular details of pilus biogenesis at the usher, and protein secretion across the outer membrane in general, are unclear. We studied the structure and oligomeric state of PapC by gel filtration, dynamic light scattering, and electron microscopy and image analysis. Two-dimensional crystals of wild-type PapC and a C-terminal deletion mutant of PapC were produced by reconstituting detergent purified usher into E.coli lipids. PapC formed a dimer both in detergent solution and in the phospholipid bilayer. Cryo-electron microscopy revealed that the usher forms a twin-pore complex. Removal of the C-terminal domain did not change the basic shape of the PapC molecule, but altered the dimeric association of the usher, suggesting that the C terminus forms part of the dimerization interface. The overall molecular size (11 nm), pore size (2 nm), and twin-pore configuration of PapC resemble that of the Tom40 complex, a mitochondrial outer membrane protein translocase.

Cell Membrane↗

PubMed2004

Evidence for conservation of architecture and physical properties of Omp85-like proteins throughout evolution.

Omp85-like proteins represent a family of proteins involved in protein translocation, and they are present in all domains of life, except archaea. In eukaryotes, Omp85-like proteins have been demonstrated to form tetrameric pore-forming complexes that interact directly with their substrate proteins. Studies performed with bacterial Omp85-like proteins have demonstrated pore-forming activity but no evidence of multimerization. In this article, we characterize the Haemophilus influenzae HMW1B protein, an Omp85-like protein that has been demonstrated to be critical for secretion of the H. influenzae HMW1 adhesin. Analysis of purified protein by biochemical and electron microscopic techniques revealed that HMW1B forms a tetramer. Examination using liposome-swelling assays demonstrated that HMW1B has pore-forming activity, with a pore size of approximately equal to 2.7 nm. Far-Western blot analysis established that HMW1B interacts with the N terminus of HMW1. These results provide evidence that a bacterial Omp85-like protein forms a tetramer and interacts directly with a substrate protein, suggesting that the architecture and physical properties of Omp85-like proteins have been conserved throughout evolution.

Bacterial Outer Membrane Proteins↗

PubMed2004

The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography.

The structure of epothilone A, bound to alpha,beta-tubulin in zinc-stabilized sheets, was determined by a combination of electron crystallography at 2.89 angstrom resolution and nuclear magnetic resonance-based conformational analysis. The complex explains both the broad-based epothilone structure-activity relationship and the known mutational resistance profile. Comparison with Taxol shows that the longstanding expectation of a common pharmacophore is not met, because each ligand exploits the tubulin-binding pocket in a unique and independent manner.

Binding Sites↗

PubMed2004

Oligomeric state of the Escherichia coli metal transporter YiiP.

YiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173-17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-beta-d-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0-68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 A revealed a pseudo-2-fold symmetry within a molecular boundary of approximately 75 x 40 A, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed.

Calibration↗