The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all known eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of the substrate–translocation channel leading to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γ-S-bound human proteasome, in which the CP gates are asymmetrically open, visualized by cryo-EM at near-atomic resolutions. At least four nucleotides are bound to the AAA-ATPase ring in these open-gate states. Variation in nucleotide binding gives rise to an axial movement of the pore loops narrowing the substrate-translation channel, which exhibit remarkable structural transitions between the spiral-staircase and saddle-shaped-circle topologies. Gate opening in the CP is thus regulated by nucleotide-driven conformational changes of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the human proteasome holoenzyme.

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Background: EpCAM or CD133 has been used as the tumor initiating cells (TICs) marker in hepatocellular carcinoma (HCC). We investigated whether cells expressing with both EpCAM and CD133 surface marker were more representative for TICs in hepatocellular carcinoma Huh7 cells.Methods: Four different phenotypes of CD133+EpCAM+, CD133+EpCAM-, CD133-EpCAM+ and CD133-EpCAM- in Huh7 cells were sorted by flow cytometry. Then cell differentiation, self-renewal, drug-resistance, spheroid formation and the levels of stem cell-related genes were detected to compare the characteristics of TICs. The ability of tumorigenicity was measured in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to verify TICs.Results: CD133+EpCAM+ cells have many characteristics of TICs in Huh7 cells compared with CD133+EpCAM-, CD133-EpCAM+, CD133-EpCAM- cells, including enrichment in side population cells, higher differentiation capacity, increased colony-formation ability, preferential expression of stem cell-related genes, appearance of drug-resistant to some chemotherapeutics, more spheroid formation of culture cells and stronger tumorigenicity in NOD/SCID mice.Conclusion: CD133+EpCAM+ phenotype is precisely represented TICs in Huh7 cells. It might be useful for studying biology mechanism of TICs in hepatocellular carcinoma and screening new targets for cancer therapy.

Bacterial chemoreceptors mediate chemotactic responses to diverse stimuli. Here, by using an integrated in silico, in vitro, and in vivo approach, we screened a large compound library and found eight novel chemoeffectors for the Escherichia coli chemoreceptor Tar. Six of the eight new Tar binding compounds induce attractant responses, and two of them function as antagonists that can bind Tar without inducing downstream signaling. Comparison between the antagonist and attractant binding patterns suggests that the key interactions for chemotaxis signaling are mediated by the hydrogen bonds formed between a donor group in the attractant and the main-chain carbonyls (Y149 and/or Q152) on the α4 helix of Tar. This molecular insight for signaling is verified by converting an antagonist to an attractant when introducing an N-H group into the antagonist to restore the hydrogen bond. Similar signal triggering effect by an O-H group is also confirmed. Our study suggests that the Tar chemoeffector binding pocket may be separated into two functional regions: region I mainly contributes to binding and region II contributes to both binding and signaling. This scenario of binding and signaling suggests that Tar may be rationally designed to respond to a nonnative ligand by altering key residues in region I to strengthen binding with the novel ligand while maintaining the key interactions in region II for signaling. Following this strategy, we have successfully redesigned Tar to respond to L-arginine, a basic amino acid that does not have chemotactic effect for WT Tar, by two site-specific mutations (R69′E and R73′E).T wo-component signaling pathways are ubiquitous in bacteria. They enable the cells to recognize and respond to different environmental stimuli (1). The control network of bacterial chemotaxis uses such a two-component system to sense the extracellular chemoeffector concentrations (2, 3). Chemoreceptors are the key upstream sensory components in the chemotaxis signaling pathway. They directly interact with specific extracellular chemoeffectors and transfer environmental information to the downstream response regulator, which ultimately controls the cell's motility (4, 5).Tar is one of the major chemoreceptors found in Escherichia coli and Salmonella enterica serovar Typhimurium (6). Attractant and repellent molecules that can induce chemotactic responses of the cells by interacting with Tar were studied (7). In addition to these two types of chemoeffectors, antagonist molecules that can directly bind to chemoreceptors without generating chemotactic responses should also exist. For example, antagonists for the sensor kinase TodS were found in the TodS/TodT two-component system (8). However, so far, antagonist molecules that function by directly binding to E. coli chemoreceptors have not been reported.Much progress has been made in understanding the structural basis of chemoreceptor signaling. Crystal structures show that each monomer (in the Tar homodimer) contains a four-helix bundle (helices α1-α4) structure, of w...

Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new β-strand, nestling into two other β-strands. Simultaneously, the apparent T m of the autoprocessed complex increases 20°C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.zymogen conversion | zinc metalloproteases inhibition | intramolecular chaperone T he thermolysin (M4) family is comprised of a large number of zinc metalloproteases in the subclan MA(E), most of which have similar sequences and domain structures (1). The representative member of this family is thermolysin (EC 3.4.24.27), a 34.6-kDa thermostable metalloprotease secreted by Bacillus thermoproteolyticus (2). The amino acid sequence and the threedimensional structure of thermolysin were first determined in 1972 (3, 4). Since then, thermolysin and thermolysin-like proteases (TLPs) have been used as models in studying zinc metalloproteases (5, 6).TLPs are widely distributed in many species of microorganisms, and many TLPs are regarded as key pathogenesis factors that are responsible for some severe bacterial infections (7). For example, λ-toxin from Clostridium perfringens can degrade various human immune defense proteins (8). Vibriolysin from Vibrio vulnificus (9) and pseudolysin from Pseudomonas aeruginosa (10) can be lethally blood-poisonous to humans. Therefore, understanding the catalytic and maturation mechanisms of TLPs is very important for developing therapeutics to treat such infectious diseases.TLPs are synthesized as a precursor with a propeptide. TLPs are divided into two distinct groups according to the primary structure of their propeptides. Less than 30% of the total TLPs have short propeptides (∼50 residues), and more than 70% of TLPs have long propeptides (∼200 residues) (11, 12). The long propeptide in TLPs contains two distinguished regions of conservation: an FTP (fungalysin/thermolysin propeptide) domain and a PepSY [peptidase propeptide and YPEB (a protein encoded by Bacillus subtilis ypeB gene)] domain. To date, the PepSY domain has been found not only in TLPs, but also in many nonpeptidase proteins, wherein the presence of this domain may prevent detrimental protease ...